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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.

Glaesserella parasuis (G. parasuis) has been one of the bacteria affecting the large-scale swine industry. Lack of an effective vaccine has limited control of the disease, which has an effect on prevalence. In order to improve the cross-protection of vaccines, development on subunit vaccines has become a hot spot. In this study, we firstly cloned the lpxC and gmhA genes from G. parasuis serotype 13 isolates, and expressed and purified their proteins. The results showed that LpxC and GmhA can stimulate mice to produce IgG antibodies. Through testing the cytokine levels of interleukin 4 (IL-4), IL-10 and interferon-γ (IFN-γ), it is found that recombinant GmhA, the mixed LpxC and GmhA can stimulate the body to produce Th1 and Th2 immune responses, while recombinant LpxC and inactivated bacteria can only produce Th2 immune responses. On the protection rate for mice, recombinant LpxC, GmhA and the mixture of LpxC and GmhA can provide 50%, 50% and 60% protection for lethal dose of G. parasuis infection, respectively. The partial protection achieved by the recombinant LpxC and GmhA supports their potential as novel vaccine candidate antigens against G. parasuis.

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.

A pharmacokinetic/pharmacodynamic (PK/PD) model was developed to optimize the dosing regimen of enrofloxacin (EN) against Glaesserella parasuis in pigs. EN (2.5 mg/kg) was administered intramuscularly to eight healthy pigs and eight pigs that were experimentally infected with G. parasuis SW124. Blood samples were collected at predetermined time points. Plasma EN concentrations were determined, and the main PK parameters were estimated. The PD of EN against G. parasuis SW124 was also investigated in vitro and ex vivo. The dynamic behaviour of EN in pigs was consistent with a one-compartment model. Significant differences were observed between healthy and infected pigs in the area under the curve (AUC) (3.58 ± 0.94 and 5.39 ± 1.01 μg h/ml, respectively) and the systemic clearance (CL) (736.32 ± 171.46 and 479.36 ± 96.81 ml/h/kg, respectively), suggesting that the pathogenicity of G. parasuis SW124 to pigs might alter the PK profile of EN, and therefore should be considered in dose optimization. Both the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were 0.125 μg/ml in tryptone soya broth (TSB) medium or plasma. The mutant prevention concentration (MPC) was 0.6 μg/ml. EN inhibited or killed G. parasuis SW124 in a concentration-dependent manner. The targeted endpoints of AUC

The pharmacokinetics of gamithromycin were evaluated in 26 male castrated and female crossbred swine administered gamithromycin 15% w/v (Zactran®, Boehringer Ingelheim) intravenously at 6 mg/kg bodyweight or intramuscularly at 3, 6 or 12 mg/kg bodyweight. Blood samples were collected up to Day 10 to establish the plasma profile of gamithromycin, bioavailability and dose proportionality. When administered by intramuscular injection at 6 mg/kg BWT, pharmacokinetic parameters were as follows: area under the curve until last quantifiable plasma concentration, 5.13 ± 0.957 µg*hours/ml; maximum plasma concentration, 960 ± 153 ng/ml at 5 to 15 min; terminal half‐life of 94.1 ± 20.4 hr. Absolute bioavailability was 92.2%. Increase in systemic exposure was proportional to the gamithromycin dose level over the range 3–12 mg/kg BWT. No gender‐related statistically significant difference in exposure was observed. For clinical evaluation of Zactran® against swine respiratory disease, 305 pigs from six commercial farms in three countries in Europe with signs associated with Actinobacillus pleuropneumoniae and/or Haemophilus parasuis and/or Pasteurella multocida and/or Bordetella bronchiseptica were used. At each site, animals were treated once in a 1:1 ratio with a single intramuscular dose of Zactran® (6 mg gamithromycin/kg bodyweight) or Zuprevo® (4% w/v tildipirosin at 4 mg/kg bodyweight; MSD Animal Health) at the recommended dose respectively. Animals were observed and scored daily for 10 consecutive days for signs of swine respiratory disease (depression, respiration and rectal temperature), and animals presenting signs of clinical swine respiratory disease (Depression Score 3 and/or Respiratory Score 3 associated with Rectal Temperature > 40.0°C) were removed from the study and considered as treatment failure. Animals which remained in the study were individually assessed for ʽtreatment successʼ or ʽtreatment failureʼ (Depression Score ≥ 1 and Rectal Temperature > 40.0°C or Respiratory Score ≥ 1 and Rectal Temperature > 40.0°C). Using a non‐inferiority hypothesis test (non‐inferiority margin = 0.10), the proportion of treatment successes in the Zactran® group (97%) was equivalent to or better than that in the Zuprevo® group (93%)., Intramuscular injection of gamithromycin 15% w/v in swine at 6 mg/kg is characterized by rapid absorption and distribution to tissues. Clinical evaluation in an European field study incorporating 305 swine has shown safe and efficacious treatment for swine with bacterial respiratory disease.

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., 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 infB gene.

Background Gamithromycin is a macrolide approved for the treatment of bovine and swine respiratory diseases. Our study aims to establish the clinical breakpoint and optimum dose regimen for gamithromycin against Haemophilus parasuis in piglets. Results Gamithromycin was well absorbed and fully bioavailable (87.2–101%) after intramuscular and subcutaneous administrations. The MICs of gamithromycin for 192 clinical H. parasuis isolates ranged from 0.008 to 128 mg/L and the epidemiological cutoff (ECOFF) was calculated as 1.0 mg/L. A large potentiation effect of serum on in vitro susceptibility of gamithromycin was observed for H. parasuis, with broth/serum ratios of 8.93 for MICs and 4.46 for MBCs, respectively. The postantibiotic effects were 1.5 h (1 × MIC) and 2.4 h (4 × MIC), and the postantibiotic sub-MIC effects ranged from 2.7 to 4.3 h. Gamithromycin had rapid and concentration-dependent killing against H. parasuis, and the AUC24h/MIC ratio correlated well with ex vivo efficacy (R2 = 0.97). The AUC24h/MIC targets in serum associated with bacteriostatic, bactericidal and eradication activities were 15.8, 30.3 and 41.2, respectively. The PK/PD-based population dose prediction indicated a probability of target attainment (PTA) for the current marketed dose (6 mg/kg) of 88.9% against H. parasuis. The calculated gamithromycin dose for a PTA ≥ 90% was 6.55 mg/kg. Based on Monte Carlo simulations, the PK/PD cutoff (COPD) was determined to be 0.25 mg/L. Conclusion The determined cutoffs and PK/PD-based dose prediction will be of great importance in gamithromycin resistance surveillance and serve as an important step in the establishment of optimum dose regimen and clinical breakpoints.

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.

Glaesserella parasuis (G. parasuis) is a commensal bacterium in the upper respiratory tract of pigs that can also cause the swine Glässer disease, which induces an intensive inflammatory response and results in significant economic losses to the swine industry worldwide. G. parasuis can cause disease through infection of the respiratory tract, resulting in systemic infection, but the mechanism is largely unknown. Recently we showed that Glaesserella parasuis serotype 4 (GPS4) increased swine tracheal epithelial barrier permeability, resulting in easier bacterial translocation. Tight junction proteins (TJ) play a crucial role in maintaining the integrity and impermeability of the epithelial barrier. GPS4 decreased the expression of the TJ ZO-1 and occludin in swine tracheal epithelial cells (STEC). Furthermore, the proinflammatory cytokines IL-6, IL-8 and TNF-α were significantly upregulated in GPS4-infected STEC, and both the MAPK and NF-κB signaling pathways were activated and contributed to the expression of TNF-α. We demonstrate that the production of proinflammatory cytokines, especially TNF-α, during GPS4 infection was involved in barrier dysfunction. Additionally, animal challenge experiments confirmed that GPS4 infection downregulated TJ in the lungs of piglets and induced a severe inflammatory response. In general, G. parasuis infection downregulated the expression of TJ and induced massive secretion of proinflammatory cytokines, resulting in epithelial barrier disruption and favoring bacterial infection. This study allowed us to better understand the mechanism by which G. parasuis crosses the respiratory tract of pigs.

In our previous study, the outer membrane protein P2 of Glaesserella parasuis (G. parasuis) induced the production of interleukin-17A (IL-17A) in host cells. However, how the G. parasuis infection produces IL-17A in the host is unknown. In this study, at 48 hpi in piglets, the proportion of Th17 cells in the peripheral blood significantly increased as determined by flow cytometry, but the overall proportion was low. IL-17 mRNA and protein levels significantly increased in the lungs and spleens of infected piglets. Histopathological examination staining, immunohistochemistry and viability counts, revealed that IL-17 expression might be positively correlated with bacterial content and the degree of pathological injury in lung and spleen tissues. Furthermore, when infected with the G. parasuis Nagasaki strain compared with avirulent strain C5, the primary porcine alveolar macrophages (PAMs) significantly produced IL-17 in a time-dose dependent manner. In addition, we demonstrated that PKC, MEK and NF-κB signaling pathways were essential for G. parasuis-induced IL-17 production because the addition of corresponding inhibitors dramatically reduced IL-17 mRNA production. The phosphorylation levels of PKC, ERK1/2 and IκB in G. parasuis-infected primary PAMs also increased significantly as the infection dose increased as assessed by western blotting. Thus, these finding imply that IL-17 induced by G. parasuis infection is dependent on activation of PKC-ERK/MAPK and IκB/NF-κB signaling pathways.

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.

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.

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., It is a comprehensive epidemiological survey of Mycoplasma hyopneumoniae (MHP), Porcine circovirus type 2 (PCV‐2) and Haemophilus parasuis (HPS) infection in pig in Shanxi of China. 2. The meteorological factors and geographic area might be important variables affecting the transmission of porcine respiratory pathogens. The study suggested that interactions between pathogens might increase the severity of porcine respiratory disease outcomes.

Glaesserella parasuis strains were characterized by serotyping PCR, vtaA virulence marker Leader Sequence (LS)-PCR, clinical significance, and geographic region. Overall, the serovars 4, 5/12, 7, 1, and 13 were the most commonly detected. Serovars of greatest clinical relevance were systemic isolates that had a higher probability of being serovar 5/12, 13, or 7. In comparison, pulmonary isolates had a higher likelihood of being serovars 2, 4, 7, or 14. Serovars 5/12 and 13 have previously been considered disease-associated, but this study agrees with other recent studies showing that serovar 7 is indeed associated with systemic G. parasuis disease. Serovar 4 strains illustrated how isolates can have varying degrees of virulence and be obtained from pulmonary, systemic, or nasal sites. Serovars 8, 9, 15, and 10 were predominantly obtained from nasal samples, which indicates a limited clinical significance of these serovars. Additionally, most internal G. parasuis isolates were classified as virulent by LS-PCR and were disease-associated isolates, including serovars 1, 2, 4, 5/12, 7, 13, and 14. Isolates from the nasal cavity, including serovars 6, 9, 10, 11, and 15, were classified as non-virulent by LS-PCR. In conclusion, the distribution of G. parasuis serovars remains constant, with few serovars representing most of the strains isolated from affected pigs. Moreover, it was confirmed that the LS-PCR can be used for G. parasuis virulence prediction of field strains worldwide.

Glässer’s disease is caused by the agent Glaesserella parasuis and is difficult to prevent and control. Candidate screening for subunit vaccines contributes to the prevention of this disease. Therefore, in this study, the inactivated G. parasuis reference serovar 5 strain (G. parasuis-5) was used to generate specific monoclonal antibodies (mAbs) to screen subunit vaccine candidates. Six mAbs (1A12, 3E3, 4C6, 2D1, 3E6, and 4B2) were screened, and they all reacted with the G. parasuis serovar 5 strain according to laser confocal microscopy and flow cytometry (FCM). Indirect enzyme-linked immunosorbent assay (ELISA) showed that one mAb 2D1, can react with all 15 reference serovars of G. parasuis. Protein mass spectrometry and Western blot analysis demonstrated that mAb 2D1 specifically reacts with Fe (3+) ABC transporter substrate-binding protein. A complement killing assay found that the colony numbers of bacteria were significantly reduced in the G. parasuis-5 group incubated with mAb 2D1 (p G. parasuis (p p G. parasuis-5. Our study found one mAb that can be used to prevent and control G. parasuis infection in vivo and in vitro, which may suggest that Fe (3+) ABC transporter substrate-binding protein is an immunodominant antigen and a promising candidate for subunit vaccine development.

Glaesserella parasuis can cause serious systemic disease (Glasser's disease) that is characterized by fibrinous polyserositis, polyarthritis and meningitis. cAMP receptor protein (CRP) is among the well studied global regulator proteins which could modulate the virulence of many pathogenic bacteria. Our previous study showed that the crp gene was involved in the regulation of growth rate, biofilm formation, stress tolerance, serum resistance, and iron utilization in G. parasuis. However, whether the crp gene could regulate the virulence of G. parasuis has not been analyzed previously. In this study, it was observed that the crp gene in G. parasuis serovar 5 (HPS5) was involved in regulating the adhesion and invasion abilities on iPAM cells, and the mRNA expression of various virulence-related factors. It also possessed the ability to induce the mRNA expression of pro-inflammatory cytokines (IL-1α, IL-1β, IL-6, IL-8 and TNF-α), promoted the activation of the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways in porcine kidney epithelial (PK-15) and immortalized swine pulmonary alveolar macrophage (iPAM) cells, and contributed to the pathogenicity and organs colonization in mice. As compared with the wild type, both the expression of virulence-related factors in the crp mutant strain and its ability to induce the mRNA expression of pro-inflammatory cytokines, as well as the expression of phospho-p65 and phospho-p38 in PK-15 and iPAM cells was reduced significantly. Furthermore, it also found that the virulence of crp mutant was significantly reduced as compared with the wild type. However, the abilities of adherence and invasion on iPAM cell of Δcrp strain was noted to be significantly enhanced as compared with the wild type. These results suggested that the crp gene deletion could effectively attenuate the virulence of G. parasuis, and crp gene may act as an important potential target for the formulation of a novel vaccine against G. parasuis.

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.

Haemophilus parasuis is part of the microbiota of the upper respiratory tract in swine. However, virulent strains can cause a systemic disease known as Glässer’s disease. Several virulence factors have been described in H. parasuis including the virulence-associated trimeric autotransporters (VtaAs). VtaA2 is up-regulated during infection and is only found in virulent strains. In order to determine its biological function, the vtaA2 gene was cloned with its native promotor region in pACYC184, and the transformed Escherichia coli was used to perform functional in vitro assays. VtaA2 was found to have a role in attachment to plastic, mucin, BSA, fibronectin and collagen. As other VtaAs from H. parasuis, the passenger domain of VtaA2 contains collagen domains. In order to examine the contribution of the collagen repeats to VtaA2 function, a recombinant vtaA2 without the central collagen domains was obtained and named vtaA2OL. VtaA2OL showed similar capacity than VtaA2 to adhere to plastic, mucin, BSA, fibronectin and plasma but a reduced capacity to adhere to collagen, suggesting that the collagen domains of VtaA2 are involved in collagen attachment. No function in cell adhesion and invasion to epithelial alveolar cell line A549 or unspecific binding to primary alveolar macrophages was found. Likewise VtaA2 had no role in serum or phagocytosis resistance. We propose that VtaA2 mediates adherence to the host by binding to the mucin, found in the upper respiratory tract mucus, and to the extracellular matrix proteins, present in the connective tissue of systemic sites, such as the serosa.

Glaesserella (formerly Haemophilus) parasuis causes Glässer’s disease, which results in high economic loss in the swine industry. To understand the polymicrobial interactions of G. parasuis and the nasal microbiota, the statistical association patterns of nasal colonizing bacteria with virulent and non-virulent strains of G. parasuis were studied accounting for the farm management practices as potential risk factors for the occurrence of Glässer’s disease. The nasal microbiota from 51 weaned-piglets from four farms with Glässer’s disease and three farms with no respiratory diseases was previously characterized and included in this study. The presence of virulent and/or non-virulent G. parasuis strains in the nasal cavities was determined in order to establish the potential association with other members of the nasal microbiota. Multivariate logistic and linear regression models were performed among the various members of nasal microbiota and G. parasuis. The multi-site production system and disease presence in the farm were both significantly associated with the presence of G. parasuis virulent strains in the nose of the piglets. Differential bacterial associations were observed with virulent or non-virulent G. parasuis. Chitinophagaceae, Corynebacteriaceae and Corynebacterium were positively associated with the virulent G. parasuis strains, while Enterobacteriaceae, Peptostreptococcaceae, Clostridium XI, and Escherichia/Shigella were negatively associated with virulent G. parasuis. On the other hand, Flavobacteriaceae, Planobacterium, and Phascolarctobacterium were positively associated with the non-virulent G. parasuis strains, while Rikenellaceae, Enterococcaceae, Odoribacter, and Corynebacterium were negatively associated with non-virulent G. parasuis. In conclusion, the nasal microbiota communities showed variations in the association with the G. parasuis strains type.

Lipooligosaccharides (LOSs) are virulence determinants of Glaesserella parasuis, a pathogen of the respiratory tract of pigs. We previously reported that disruption of the galU or galE gene in G. parasuis results in increased sensitivity to porcine serum, indicating that the galactose catabolism pathway is required for polysaccharide formation in G. parasuis. Here, we evaluated the role of the HAPS_0849 gene in LOS synthesis. The G. parasuis SC096 HAPS_0849 mutant produced a highly truncated LOS molecule, although a small fraction of intact LOS was still observed, and this mutant was found to be more sensitive to serum than the parental strain. HAPS_0849 was overexpressed and purified for biochemical assays, and this protein exhibited phosphoglucomutase (PGM) activity. Heterologous expression of a pgm gene from Escherichia coli in the HAPS_0849 mutant led to restoration of the wild-type LOS glycoform, further demonstrating the PGM function of HAPS_0849 in G. parasuis. The autoagglutination and biofilm formation ability of this strain were also investigated. Disruption of HAPS_0849 led to an increased tendency to autoagglutinate and form more biofilms, and these enhanced phenotypes were observed in the absence of glucose. In addition, LOSs from HAPS_0849, galU and lgtB mutants had similar truncated glycoforms, while LOSs from the galE and lex-1 mutants exhibited another type of defective LOS pattern. These findings imply that HAPS_0849 may function upstream of GalU in the generation of glucose 1-phosphate. In conclusion, our results preliminarily described the functions of HAPS_0849 in G. parasuis, and this gene was partially required for LOS synthesis.

Glaesserella parasuis (G. parasuis) causes porcine vascular inflammation and damage. Baicalin is reported to have antioxidant and anti-inflammatory functions. However, whether baicalin protects piglets against G. parasuis challenge and the potential protective mechanism have not been investigated. Therefore, in this study, we comprehensively examined the protective efficacy of baicalin in piglets challenged with G. parasuis and the possible protective mechanism. Our results show that baicalin attenuated the release of the inflammation-related cytokines interleukin (IL) 1β, IL6, IL8, IL10, and tumour necrosis factor α (TNF-α) and reduced high mobility group box 1 (HMGB1) production and cell apoptosis in piglets infected with G. parasuis. Baicalin also inhibited the activation of the mitogen-activated protein kinase (MAPK) signalling pathway and protected piglets against G. parasuis challenge. Taken together, our data suggest that baicalin could protect piglets from G. parasuis by reducing HMGB1 release, attenuating cell apoptosis, and inhibiting MAPK signalling activation, thereby alleviating the inflammatory response induced by the bacteria. Our results suggest that baicalin has utility as a novel therapeutic drug to control G. parasuis infection.

Glaesserella parasuis (G. parasuis) is an important pathogenic bacterium that can cause Glasser's disease, and it has resulted in tremendous economic losses to the global swine industry. The intensive pulmonary inflammatory response caused by G. parasuis infection is the main cause of lung injury and death in pigs. However, the exact mechanism by which it causes severe pulmonary inflammation is not fully understood yet. In this study, severe pneumonia was observed in piglets infected with G. parasuis; and an infection cell model was established using porcine alveolar macrophages cell line 3D4/21, which was determined to be susceptible to G. parasuis infection in vitro. G. parasuis infection of 3D4/21 cells induced upregulation of proinflammatory cytokines TNF-α, IL-1β, IL-18 and production of intracellular reactive oxygen species (ROS). The expression of IL-1β related to activation of the NLRP3 inflammasome signaling pathway, which had not been shown before in G. parasuis infection. Furthermore, it was first found that release of intracellular ROS, which was mediated by NADPH oxidase in 3D4/21 cells, was found crucial for the activation of the NLRP3 signaling pathway and promoted the expression of proinflammatory cytokines, such as TNF-α and IL-1. In general, this study explored the specific mechanism of severe pulmonary inflammation caused by G. parasuis infection, and provides a foundation for further elucidating the pathogenic mechanism of G. parasuis.

Glaesserella (Haemophilus) parasuis is a part of the microbiota of healthy pigs and also causes the systemic condition called Glasser's disease. G. parasuis is categorized by it capsular polysaccharide into 15 serovars. Because of the serovar and strain specific immunity generated by whole cell vaccines and the rapid onset of disease, G. parasuis has been difficult to control in the swine industry. This report investigated the protection afforded by the use of two serovar 5 isolates (Nagasaki and HS069) as whole cell, killed bacterins against homologous challenge and heterologous challenge with the serovar 1 strain 12939 to better understand bacterin generated immunity. Both bacterins induced a high antibody titer to the vaccine strain and the heterologous challenge strain. Protection was seen with both bacterins against homologous challenge; however, after heterologous challenge, the HS069 bacterin provided complete protection and all Nagasaki bacterin vaccinated animals succumbed to disease. The difference in protection appears to be due to differences in antibody specificity and the capacity of induced antibody to fix complement and opsonize G. parasuis, as shown by Western blotting and functional assays. This report shows the importance of strain selection when developing bacterin vaccines, as some strains are better able to generate heterologous protection. The difference in protection seen here can also be utilized to detect proteins of interest for subunit vaccine development.

Haemophilus parasuis (H. parasuis) is a conditional pathogen with the ability to form biofilms which can lead to ineffective drug treatment and severe chronic infections resulting in significant economic losses to the pig industry. Currently, knowledge of biofilm formation by H. parasuis is not well developed. The objective of this study was to investigate the three-dimensional morphology of biofilms and perform transcriptomic analysis on H. parasuis cells in biofilm versus planktonic forms. The results showed that proteins and DNA accounted for a large proportion of the H. parasuis biofilm extracellular matrix. Here, we have traced the entire biofilm formation process of H. parasuis from beginning to end for the first time. These biofilms grew rapidly in the first 48 h and became stable at 60 h. According to GO and KEGG analysis, the differentially expressed genes (DEG) artM, artQ, ssrS, pflA and HutX were implicated as being involved in bacterial colonisation and adhesion; these are the most likely genes to affect biofilm formation. Most functional gene enrichments were of those involved in metabolic pathways, biosynthesis of secondary metabolites, ATP-binding cassette (ABC) transporters, and starch and sucrose metabolism. Thus, in the present pilot study, the composition and characteristics of these biofilms were explored, and the genes related to biofilm formation were screened for. This research lays the foundation for further studies on mechanisms regulating biofilm formation, in order to find new drug targets and develop new therapeutic drugs against H. parasuis.

Glaesserella parasuis (G. parasuis) is a respiratory pathogen of swine and the etiological agent of Glasser's disease. Although the emergence of multidrug-resistant (MDR) G. parasuis is a critical problem in the swine industry, there are few publications on the genetic basis of antimicrobial resistance of G. parasuis. In this study, comparative genome analyses were used to identify genomic differences between two phenotypically distinct isolates, an MDR isolate (HPS-1) and a susceptible isolate (HPS-2), from diseased swines in China. These isolates were both serovar 4, which is predominant in cases of Glasser's disease and is the most prevalent serovar in China. Based on clusters of orthologous group (COG) annotations, genes assigned to the extracellular structure category were only detected in HPS-1 and genes related to cell motility were more abundant in HPS-1 than in HPS-2. A comparative genomic analysis showed that these two isolates are closely related, although there was a large-scale genomic rearrangement. Eighteen percent of the genome consisted of strain-specific accessory genes of HPS-1. Notably, only the two genes aac(6')-Ie-aph(2'')-Ia and blaROB-1 on a plasmid were specific to HPS-1. We also detected 30,599 single nucleotide polymorphisms (SNPs), including nonsynonymous SNPs in the aminoglycoside resistance gene aph(3'')-Ib, the fusidic acid resistance gene fusA, and the two rifampicin resistance genes rpoC and rpoB in HPS-1. These findings improve our understanding of the differences between MDR and susceptible isolates and will aid the development of treatment strategies to decrease the prevalence and disease burden caused by G. parasuis.

Haemophilus parasuis is the main agent of Glasser's disease, which causes substantial losses in pig production. However, the pathogenic mechanism and virulence factors of H. parasuis have not been fully determined. In this study, berberine is shown to have a good therapeutic effect in vivo against H. parasuis; the minimal inhibitory concentration (MIC) in vitro was 2 μg/mL. Berberine inhibited H. parasuis adhesion to and invasion of PK-15 pig kidney cells. Proteomics studies of H. parasuis after berberine treatment identified a total of 97 differentially-expressed proteins; 35 upregulated and 62 downregulated. Bioinformatics analysis showed that berberine may inhibit the growth of H. parasuis by affecting outer membrane proteins, transferrins, and energy metabolism. This study provides a basis for the development of new antibacterial agents.

BackgroundGlaesserella parasuis, the causative agent of Glӓsser’s disease, is widespread in swine globally resulting in significant economic losses to the swine industry. Prevention of Glӓsser’s disease in pigs has been plagued with an inability to design broadly protective vaccines, as many bacterin based platforms generate serovar or strain specific immunity. Subunit vaccines are of interest to provide protective immunity to multiple strains ofG. parasuis. Selected proteins for subunit vaccination should be widespread, highly conserved, and surface exposed.ResultsTwo candidate proteins for subunit vaccination (RlpB and VacJ) againstG. parasuiswere identified using random mutagenesis and an in vitro organ culture system. Pigs were vaccinated with recombinant RlpB and VacJ, outer membrane proteins with important contributions to cellular function and viability. Though high antibody titers to the recombinant proteins and increased interferon-γ producing cells were found in subunit vaccinated animals, the pigs were not protected from developing systemic disease.ConclusionsIt appears there may be insufficient RlpB and VacJ exposed on the bacterial surface for antibody to bind, preventing high RlpB and VacJ specific antibody titers from protecting animals fromG. parasuis. Additionally, this work confirms the importance of utilizing the natural host species when assessing the efficacy of vaccine candidates.

Antimicrobials have been commonly used to control bacterial diseases in farm animals. The efficacy of these drugs deterred the development of other control measures, such as vaccines, which are currently getting more attention due to the increased concern about antimicrobial resistance. Glässer’s disease is caused by Glaesserella (Haemophilus) parasuis and affects pork production around the world. Balance between colonization and immunity seems to be essential in disease control. Reduction in antimicrobial use in veterinary medicine requires the implementation of preventive measures, based on alternative tools such as vaccination and other strategies to guarantee a beneficial microbial colonization of the animals. The present review summarizes and discusses the current knowledge on diagnosis and control of Glässer’s disease, including prospects on alternatives to antimicrobials. info:eu-repo/semantics/publishedVersion

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.

Porcine reproductive and respiratory syndrome virus (PRRSV) predisposes pigs to secondary bacterial infection caused by Haemophilus parasuis. The aim of the present study was to analyse the immune response of monocyte-derived macrophages (MDMs), serving as a model of macrophages accumulating at the site of inflammation. The second part of the study was focused on the role of IFNα in the production of inflammatory cytokines in co-infected MDMs. Concurrent infection with PRRSV and H. parasuis decreased gene expression of pro-inflammatory cytokines (IL-1β, IL-8) in MDMs in comparison with MDMs infected with PRRSV or H. parasuis alone. Our data showed that MDMs express IFNα after PRRSV infection. Thereafter, we exposed cells to the experimental addition of IFNα and a subsequent infection with H. parasuis, and detected a decreased expression/production of pro-inflammatory cytokines. Thus, we assume that IFNα, produced after PRRSV infection, could affect the immune response of monocyte-derived macrophages. Down-regulation of pro-inflammatory cytokine expression in inflammatory macrophages may allow the development of secondary bacterial infections in pigs.

Outer membrane protein P2 (OmpP2) of the virulent Haemophilus (Glaesserella) parasuis has been shown to induce the release of proinflammatory cytokines. The OmpP2 protein is composed of eight or nine surface-exposed loops, but it is unclear which of them participates in the OmpP2-induced inflammatory response. In this study, we synthesized linear peptides corresponding to surface-exposed loops L1–L8 of OmpP2 from the virulent H. parasuis SC096 strain to stimulate porcine alveolar macrophages (PAMs) in vitro. We found that both L7 and L8 significantly upregulated the mRNA expression of interleukin (IL)-1α, IL-1β, IL-6, IL-8, IL-17, and IL-23 and the chemokines CCL-4 and CCL-5 in a time- and dose-dependent manner. Additionally, we constructed ompP2ΔLoop7 and ompP2ΔLoop8 mutant SC096 strains and extracted their native OmpP2 proteins to stimulate PAMs. These mutant proteins induced significantly less mRNA expression of inflammatory cytokines than SC096 OmpP2. Next, the amino acid sequences of L7 and L8 from 15 serovars of H. parasuis OmpP2 were aligned. These sequences were relatively conserved among the most virulent reference strains, suggesting that L7 and L8 are the most active peptides of the OmpP2 protein. Furthermore, L7 and L8 significantly upregulated the NF-κB and AP-1 activity levels based on luciferase reporter assays in a dose-dependent manner. Therefore, our results demonstrated that both surface-exposed loops L7 and L8 of H. parasuis OmpP2 induced the expression of proinflammatory cytokines possibly by activating the NF-κB and MAPK signalling pathways in cells infected by H. parasuis.

Three recombinant outer membrane proteins (rOmps) from the Haemophilus parasuis Nagasaki strain (serovar 5 reference strain), rOmpP2, rOmpP5 and rOmpD15, which have previously shown protection against H. parasuis infection in mice, were cloned, expressed and evaluated as vaccine antigens in colostrum-deprived pigs. When these animals were immunized with these rOmps and were later challenged intratracheally with 108 CFUs of the Nagasaki strain, no protection was seen in terms of survival, clinical signs, pathological results and recovery of H. parasuis. We hypothesized that a possible explanation for this lack of protection could be the low number of epitopes accessible to the immune system as a consequence of their poor exposure on the bacterial surface so that the immune response would not be able to protect against experimental infection by H. parasuis when a fully susceptible animal model, such as pigs, was used.

Determine if there is a link between virulence-associated genes of Haemophilus parasuis and the genotype and serovar of isolates. 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. 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. 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.

The combined antibacterial effects of tilmicosin (TIL) and florfenicol (FF) against Actinobacillus pleuropneumoniae (APP) (n = 2), Streptococcus suis (S. suis) (n = 2), and Haemophilus parasuis (HPS) (n = 2) were evaluated by chekerboard test and time-kill assays. The pharmacokinetics (PKs) of TIL- and FF-loaded hydrogenated castor oil (HCO)-solid lipid nanoparticles (SLN) were performed in healthy pigs. The results indicated that TIL and FF showed synergistic or additive antibacterial activities against APP, S. suis and HPS with the fractional inhibitory concentration (FIC) ranging from 0.375 to 0.75. The time-kill assays showed that 1/2 minimum inhibitory concentration (MIC) TIL combined with 1/2 MIC FF had a stronger ability to inhibit the growth of APP, S. suis, and HPS than 1 MIC TIL or 1 MIC FF, respectively. After oral administration, plasma TIL and FF concentrations could maintain about 0.1 μg/ml for 192 and 176 hr. The SLN prolonged the last time point with detectable concentrations (Tlast), area under the concentration–time curve (AUC0-t), elimination half-life (T½ke), and mean residence time (MRT) by 3.1, 5.6, 12.7, 3.4-fold of the active pharmaceutical ingredient (API) of TIL and 11.8, 16.5, 18.1, 12.1-fold of the API of FF, respectively. This study suggests that the TIL-FF-SLN could be a useful oral formulation for the treatment of APP, S. suis, and HPS infection in pigs.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant and economically important infectious diseases affecting swine worldwide and can predispose pigs to secondary bacterial infections caused by, e.g. Haemophilus parasuis. The aim of the presented study was to compare susceptibility of two different types of macrophages which could be in contact with both pathogens during infection with PRRS virus (PRRSV) and in co-infection with H. parasuis. Alveolar macrophages (PAMs) as resident cells provide one of the first lines of defence against microbes invading lung tissue. On the other hand, monocyte derived macrophages (MDMs) represent inflammatory cells accumulating at the site of inflammation. While PAMs were relatively resistant to cytopathogenic effect caused by PRRSV, MDMs were much more sensitive to PRRSV infection. MDMs infected with PRRSV increased expression of pro-apoptotic Bad, Bax and p53 mRNA. Increased mortality of MDMs may be also related to a higher intensity of ROS production after infection with PRRSV. In addition, MDMs (but not PAMs) infected with H. parasuis alone formed multinucleated giant cells (MGC); these cells were not observed in MDMs infected with both pathogens. Higher sensitivity of MDMs to PRRSV infection, which is associated with limited MDMs survival and restriction of MGC formation, could contribute to the development of multifactorial respiratory disease of swine. Electronic supplementary material The online version of this article (doi:10.1186/s13567-017-0433-6) contains supplementary material, which is available to authorized users.

Haemophilus parasuis (H. parasuis) is the etiological agent of swine Glasser's disease, which leads to significant economic loss in swine industry over the world. Subunit vaccine based on outer membrane protein is one of the promising choices to protect pigs against H. parasuis infection despite low immunity efficiency. In this paper, outer membrane protein 16 (Omp16) of H. parasuis encapsulated by alginate-chitosan microspheres as antigen carriers was explored for the first time in a mouse model. Our results showed that the microspheres with Omp16 induced significant higher H. parasuis-specific antibodies, and higher titers of IL-2, IL-4, and IFN-γ than those by Omp16-FIA in treated mice (p

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

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.

Background Glaesserella parasuis (G. parasuis) is an influential pathogen of the pig, which induces high morbidity and mortality in naive pig populations in the pig industry. Accurate and rapid detection of the agent is important for disease control. In this study, a simple recombinase polymerase amplification (RPA) with a Lateral flow (LF) strip (RPA-LF-GPS) was developed to detect G. parasuis. Results The RPA-LF-GPS can specifically detect G. parasuis a limit of 100 CFU from other common related pathogens causing arthritis in the pig. The RPA-LF-GPS assay can use boiled synovial fluid samples as a template with the same sensitivity as other DNA extraction methods. In the detection of clinic positive synovial fluid sample, RPA-LF-GPS is equally sensitive (98.1%) compared with that of PCR (90.4%) (P > 0.05). The whole procedure of the RPA-LF-GPS assay could be finished in 1 hour without professional equipment. Conclusions RPA-LF-GPS assay is a rapid and simple method for point-of-care diagnostic testing for G. parasuis infection.

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.

Haemophilus parasuis is a common colonizer of the upper respiratory tract of swine and frequently causes disease, especially in weaner pigs. To date, limited epidemiological data was available for H. parasuis from healthy pigs, which might be carriers of potential pathogenic strains. In this study, from September 2016 to October 2017, we investigated the prevalence and characteristics of H. parasuis from healthy pigs in China. Totally, we obtained 244 isolates from 1675 nasal samples from 6 provinces. H. parasuis isolation was more successful in weaner pigs (22.6%, 192/849), followed by finisher pigs (9.3%, 43/463), and sows (2.5%, 9/363). The most prevalent serovars were 7 (20.1%, 49/244), followed by 3 (14.8%, 36/244), 2 (14.3%, 35/244), 11 (12.7%, 31/244), 5/12 (5.7%, 14/244) and 4 (2.5%, 6/244). Bimodal or multimodal distributions of MICs were observed for most of the tested drugs, which suggested the presence of non-wild type populations. It was noted that the MIC90 values of tilmicosin (64 μg/ml) was relatively higher than that reported in previous studies. Our results suggest that: 1) potentially pathogenic serovars of H. parasuis are identified in healthy pigs, and 2) elevated MICs and presence of mechanisms of resistance not yet described for clinically important antimicrobial agents would increase the burden of disease caused by H. parasuis. info:eu-repo/semantics/acceptedVersion

Glaesserella parasuis is the causative agent of Glasser's disease in swine. Serotyping plays an essential role in prevalence investigations and in the development of vaccination strategies for the prevention of this disease. Molecular serotyping based on variation within the capsule loci of the 15 serovars is more accurate and efficient than traditional serological serotyping. To reduce the running time and facilitate ease of data interpretation, we developed a simple and rapid cycle threshold (Ct) value-based real time PCR (qPCR) method for the identification and serotyping of G. parasuis. The qPCR method distinguished between all 15 serovar reference strains of G. parasuis with efficiency values ranging between 85.5 % and 110.4 % and, R2 values > 0.98. The qPCR serotyping was evaluated using 83 clinical isolates with 43 of the isolates having been previously assigned to a serovar by the gel immuno-diffusion (GID) assay and 40 non-typeable isolates. The qPCR results of 41/43 (95.3 %) isolates were concordant with the GID assay except two isolates of serovar 12 were assigned to serovar 5. In addition, the qPCR serotyping assigned a serovar to each of the 40 non-typeable isolates. Of the 83 isolates tested to assign a serovar, a concordance rate of 98.8 % (82/83) was determined between the qPCR and the previously reported multiplex PCR of Howell et al. (2015) (including those that were either serovars 5 or 12). Despite the inability to differentiate between serovars 5 and 12, the Ct value-based qPCR serotyping represents an attractive alternative to current molecular serotyping method for G. parasuis and could be used for both epidemiological monitoring and the guidance of vaccination programs.

Glässer's disease, which is caused by Haemophilus parasuis, is a major threat to swine throughout the world. At present, the predominant method of controlling this disease is through vaccination with an inactivated vaccine, which has many limitations. For example, there is no available method to differentiate between infected and vaccinated animals. In addition, inactivated vaccines do not contain all of the virulent serovars that circulate in a region. Additionally, novel vaccines that address the shortcomings of inactivated vaccines are not available. Here, we summarize existing knowledge concerning H. parasuis vaccines, and provide some suggestions that may help to improve H. parasuis vaccines.

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

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

Haemophilus parasuis is an important swine pathogen that causes Glasser's disease, characterized by pneumonia, polyserositis and meningitis. Protection against H. parasuis infection is associated with the presence of homologous antibodies in serum. However, a H. parasuis antigen that can elicit a protective immune response against all H. parasuis strains has yet to be found. A novel immunogenic and species-specific H. parasuis protein was identified by screening H. parasuis whole cell proteins using swine convalescent sera. One protein of 52kDa was clearly immunodominant and conserved among different H. parasuis strains. This protein was further identified as an oligopeptide permease A (OppA). Because OppA elicited a specific antibody response in pigs that recovered from H. parasuis infection, we investigated its potential role in diagnostics and protective immunity. An ELISA test using recombinant OppA (rOppA) as its coating antigen was further developed and tested. H. parasuis specific antibodies to rOppA were detected in serum from convalescent pigs but not in serum from specific pathogen free (SPF) or conventional pigs. Pigs immunized with rOppA protein had robust serological responses. However, the antibodies were not protective against challenge infection. We conclude that OppA is a universal species-specific H. parasuis immunogen, and a good marker for previous systemic infection with H. parasuis.

Objective To identify genes associated with the observed antimicrobial resistance in Actinobacillus pleuropneumoniae, Haemophilus parasuis and Pasteurella multocida isolated from Australian pigs. Design Isolates with known phenotypic resistance to β-lactams, macrolides and tetracycline were screened for the presence of antimicrobial resistance genes. Procedure A total of 68 A. pleuropneumoniae, 62 H. parasuis and 20 P. multocida isolates exhibiting phenotypic antimicrobial resistance (A. pleuropneumoniae and P. multocida) or elevated minimal inhibitory concentrations (MICs) (H. parasuis) to any of the following antimicrobial agents − ampicillin, erythromycin, penicillin, tetracycline, tilmicosin and tulathromycin − were screened for a total of 19 associated antimicrobial resistance genes (ARGs) by PCR. Results The gene bla ROB-1 was found in all ampicillin- and penicillin-resistant isolates, but none harboured the bla TEM-1 gene. The tetB gene was found in 76% (74/97) of tetracycline-resistant isolates, 49/53 A. pleuropneumoniae, 17/30 H. parasuis and 8/14 P. multocida. One A. pleuropneumoniae isolate harboured the tetH gene, but none of the 97 isolates had tetA, tetC, tetD, tetE, tetL, tetM or tetO. A total of 92 isolates were screened for the presence of macrolide resistance genes. None was found to have ermA, ermB, ermC, erm42, mphE, mefA, msrA or msrE. Conclusion The current study has provided a genetic explanation for the resistance or elevated MIC of the majority of isolates of Australian porcine respiratory pathogens to ampicillin, penicillin and tetracycline. However, the macrolide resistance observed by phenotypic testing remains genetically unexplained and further studies are required.

Haemophilus parasuis is the causative agent of Glässer's disease, which causes high morbidity and mortality in piglets, leading to severe economic losses. The lack of a commercial vaccine against a broad spectrum of strains has limited the disease control. H. parasuis outer membrane proteins (OMPs) are potentially essential components for vaccine formulation. In this study, seven putative OMPs were selected from the annotated H. parasuis serovar 5 genome; they were predicted by bioinformatics and annotated as potential virulence-related factors. These proteins were cloned, expressed, and purified as His-tagged proteins. Antigenicity of the candidate proteins was assessed using Western blotting with convalescent sera against H. parasuis serovar 5. The immunogenicity of the seven OMPs was assessed in a guinea pig model. Except VacJ, all the other six recombinant proteins elicited a detectable antibody response. Antisera against four of the selected proteins effectively killed the bacteria in vitro. Three proteins (Omp26, VacJ, and HAPS_0742) were found to confer significant protection against challenge with a lethal dose of H. parasuis in a guinea pig model. The results suggest that these three proteins have a strong potential to be vaccine candidates against Glässer's disease.