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Porcine respiratory disease is a significant economic problem for the global swine industry. Haemophilus parasuis ( H. parasuis ), Streptococcus suis ( S. suis ), and Pasteurella multocida ( P. multocida ) are three important pathogenic bacteria of the swine respiratory tract. Notably, the three pathogens not only frequently manifest as mixed infections, but their striking clinical similarities also present difficulties for pig populations in terms of disease prevention and treatment. Thus, we developed a triplex real-time quantitative polymerase chain reaction (qPCR) assay based on a TaqMan probe for the detection of H. parasuis , S. suis serotype 2, and P. multocida . Primers and probes were designed to target the conserved regions of the H. parasuis OmpP2 gene, the S. suis serotype 2 gdh gene, and the P. multocida Kmt1 gene. By optimizing the reaction system and conditions, a triplex qPCR method for simultaneous detection of H. parasuis , S. suis serotype 2, and P. multocida was successfully established. The amplification efficiencies of the standard curves for all three pathogens were found to be highly similar, with values of 102.105% for H. parasuis , 105.297% for S. suis serotype 2, and 104.829% for P. multocida , and all R 2 values achieving 0.999. The specificity analysis results showed that the triplex qPCR method had a strong specificity. The sensitivity test results indicated that the limit of detection can reach 50 copies/μL for all three pathogens. Both intra- and inter-assay coefficients of variation for repeatability were below 1%. This triplex qPCR method was shown to have good specificity, sensitivity, and reproducibility. Finally, the triplex qPCR method established in this study was compared with the nested PCR as recommended by the Chinese national standard (GB/T34750-2017) for H. parasuis , the PCR as recommended by the Chinese national standard (GB/T 19915.9-2005) for S. suis serotype 2, and the PCR as recommended by the Chinese agricultural industry standard (NY/T 564-2016) for P. multocida by detecting the same clinical samples. Both methods are reasonably consistent, while the triplex qPCR assay was more sensitive. In summary, triplex qPCR serves not only as a rapid and accurate detection and early prevention method for these pathogens but also constitutes a robust tool for microbial quality control in specific pathogen-free pigs.

Swine Glasser's disease, instigated by Haemophilus parasuis ( H. parasuis ), is a significant bacterial infection that causes substantial economic losses in pig farming operations. The role of mucosal immunity is pivotal in defending against H. parasuis . This study focused on the construction of PLGA microspheres that encapsulate the outer membrane protein OMP16 from H. parasuis (PLGA-OMP16) and evaluated their immunological effectiveness in a mouse model. After being intranasally immunized twice, the PLGA-OMP16 microspheres effectively induced IgAs in saliva and nasal and lung fluids. The PLGA-OMP16 microspheres also significantly increased the number of anti H. parasuis IgGs in serum. Furthermore, the PLGA-OMP16 microspheres triggered elevated levels of IL-2, IL-4, and IFN-γ. The mice vaccinated with PLGA-OMP16 showed a significant reduction in H. parasuis burden in the spleen and lungs following bacterial challenge. These results indicate that intranasal immunization using PLGA microspheres is a promising adjuvant delivery system for vaccines targeting H. parasuis .

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&#95;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&#95;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.

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., Competing Interests: No potential conflict of interest relevant to this article was reported., (© Copyright 2024 Korean Society of Animal Science and Technology.)

Glaesserella parasuis, an important respiratory bacterial pathogen, causes Glässer's disease in piglets, with potential immunosuppression. We established a piglet infection model and explored the immunosuppression mechanism to improve our understanding of the host immune response to G. parasuis. Twenty piglets were randomly divided into two groups (n = 10). The infection group was intraperitoneally challenged with 2 × 10 8 CFU of G. parasuis in 2 mL TSB. The control group was intraperitoneally injected with equivalent TSB. After 72 h, the piglets were sacrificed, and spleen tissue was collected. PD-1/PD-L1 expression was determined. The splenocytes were isolated to detect CD3 + T, CD3 + CD4 + T, CD3 + CD8 + T and CD3 - CD21 + cell differentiation. Via data-independent acquisition (DIA), we compared the proteomics of healthy and infected spleen tissues. Glaesserella parasuis modified CD3 + T, CD3 + CD4 + T, CD3 + CD8 + T and CD3 - CD21 + cell differentiation and PD-1/PD-L1 expression in the spleen. The infection group had 596 proteins with significant differences in expression, of which 301 were significantly upregulated and 295 downregulated. Differentially expressed proteins (DEPs) were mainly related to immune responses. This is the first study on PD-1/PD-L1 expression in the spleen associated with immunosuppression in a piglet model to explore the protein changes related to immune responses via DIA., (© 2024. The Author(s).)

Introduction: Matrine (MT) is a potential resistance reversal agent. However, it remains unclear whether MT can reverse the resistance of Haemophilus parasuis ( H. parasuis ) to β-lactams, and, if so, by what mechanism MT works., Methods: We screened one cefaclor (CEC)-resistant strain (clinical strain C7) from eight clinical ( H. parasuis ) strains and determined the underlying resistance mechanism. Then, we investigated the reversal effect of MTon the resistance of this strain to CEC., Results and Discussion: The production of β-lactamase, overexpression of 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 (Y 103 D and L 517 R) and mrcA (A 639 V) 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhao, Yang, Deng, Li, Wang, Yi, Kuang, Xu, Li, Li, Yu and Yang.)

Background: Haemophilus parasuis (H. parasuis) is a gram-negative bacterial pathogen that causes severe infections in swine, resulting in substantial economic losses. Currently, the majority of H. parasuis detection methods are impractical for on-site application due to their reliance on large instruments or complex procedures. Thus, there is an urgent need to develop a rapid, visually detectable, and highly sensitive detection method, especially under resource-limited environments and field conditions., Results: In this study, we established a naked eye assay for highly sensitive detection by combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology. Positive samples exhibited a clear red color visible to the naked eye, while negative samples appeared blue. We achieved a remarkable sensitivity, detecting H. parasuis down to a single copy, with no cross-reactivity with other bacteria. In a mouse model, our assay detected H. parasuis infection nearly 8 h earlier than traditional PCR. Compared to qPCR, our detection results were 100 % accurate. To enhance point-of-care applicability and mitigate the risk of aerosol contamination from uncapping, we consolidated RPA and CRISPR/Cas12a cleavage into a single-tube reaction system. This integrated approach was validated with 20 clinical lung samples, yielding results consistent with those obtained from qPCR. The entire procedure, from DNA extraction to detection, was completed in 35 min., Significance: We present an RPA-CRISPR/Cas12a assay suitable for the early and resource-efficient diagnosis of H. parasuis infections. Its simplicity and visual detection are advantageous for field diagnostics, representing a substantial develpoment in the diagnosis of H. parasuis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Haemophilus 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.

Aims: Swine respiratory disease (SRD) is a major disease complex in pigs that causes severe economic losses. SRD is associated with several intrinsic and extrinsic factors such as host health status, viruses, bacteria, and environmental factors. Particularly, it is known that many pathogens are associated with SRD to date, but most of the test to detect those pathogens can be normally investigated only one pathogen while taking time and labor. Therefore, it is desirable to develop rapidly and efficiently detectable methods those pathogens to minimize the damage caused by SRD., Methods and Results: We designed a multiplex real-time RT-PCR (RT-qPCR) system to diagnose simultaneously 16 pathogens, including nine viruses and seven bacteria associated with SRD, on the basis of single qPCR and RT-qPCR assays reported in previous studies. Multiplex RT-qPCR system we designed had the same ability to single RT-qPCR without significant differences in detection sensitivity for all target pathogens at minimum to maximum genomic levels. Moreover, the primers and probes used in this system had highly specificity because the sets had not been detected pathogens other than the target and its taxonomically related pathogens. Furthermore, our data demonstrated that this system would be useful to detect a causative pathogen in the diagnosis using oral fluid from healthy pigs and lung tissue from pigs with respiratory disorders collected in the field., Conclusions: The rapid detection of infected animals from the herd using our system will contribute to infection control and prompt treatment in the field., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

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 10 4 -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.

Glaesserella parasuis is an important pathogen that causes fibrinous polyserositis, peritonitis and meningitis in pigs, leading to considerable economic losses to the swine industry worldwide. It is well established that the serine protease HtrA is closely associated with bacterial virulence, but the role of HtrA in G. parasuis pathogenesis remains largely unknown. To characterize the function of the htrA gene in G. parasuis, a ΔhtrA mutant was constructed. We found that the ΔhtrA mutant showed significant growth inhibition under heat shock and alkaline stress conditions, indicating HtrA is involved in stress tolerance and survival of G. parasuis. In addition, deletion of htrA gene resulted in decreased adherence to PIEC and PK-15 cells and increased phagocytic resistance to 3D4/2 macrophages, suggesting that htrA is essential for adherence of G. parasuis. Scanning electron microscopy revealed morphological surface changes of the ΔhtrA mutant, and transcription analysis confirmed that a number of adhesion-associated genes are downregulated, which corroborated the aforementioned phenomenon. Furthermore, G. parasuis HtrA induced a potent antibody response in piglets with Glässer's disease. These observations confirmed that the htrA gene is related to the survival and pathogenicity of G. parasuis., Competing Interests: Declaration of Competing Interest The authors declare that there are no financial or other relationships that might lead to a conflict of interest. All authors have seen and approved the manuscript., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Glässer's disease is caused by Glaesserella parasuis , a common bacterium in the upper respiratory tract of pigs. Antibiotics are widely used to control this disease. A G. parasuis isolate with amoxicillin (AMX) resistance was identified in our previous study. Outer membrane vesicles (OMVs) are naturally released by G. parasuis and contain many compounds. To reveal the underlying mechanisms associated with AMX resistance delivery, OMVs from G. parasuis were successfully isolated and identified by transmission electron microscopy. In particular, we found that β-lactamase existed in OMVs using label-free analysis, and further verified that OMVs carry β-lactamase by Western blotting. The minimal inhibitory concentration and growth rate were determined to evaluate the β-lactamase activity in G. parasuis OMVs. Moreover, the effect of different concentrations of OMVs from aHPS7 on the growth rate of AMX sensitive strains was examined. Our results further confirmed that OMVs isolated from aHPS7 contain β-lactamase, which can prevent AMX-susceptible strains from killing by hydrolyzing AMX. Our results first showed that OMVs of G. parasuis play an important role in the spread of antibiotic resistance, which seriously hampered the prevention of this disease by the delivery of OMVs in different strains.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wu, Nan, Peng, Hu, Xu, Huang and Xiao.)

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) is commonly located in the upper respiratory tract of pigs as an opportunistic pathogen. It can cause Glässer's disease, which leads to serious economic losses in the swine industry. The occurrence of the disease is often linked with the adhesion and colonization of the pathogen. The PilA pilus subunit is important for adhesion to the host, twitching motility, and biofilm formation in many bacteria. However, no research has focused on the function of PilA in G. parasuis . To further reveal the pathogenesis of G. parasuis and to search for subunit vaccine candidates, we investigated whether PilA could adhere to cells and provide immune protection. A bioinformatic analysis showed that the protein secondary structure of the G. parasuis PilA was similar to that of Haemophilus influenzae (HI). Cell adhesion, ELISA, and far-Western blotting showed that rPilA could bind porcine-derived, porcine kidney-15 (PK-15) cells, swine tracheal epithelial cells (STECs), and the extracellular matrix components fibronectin (FN) and laminin (LN). An immunogenicity analysis showed that recombinant PilA (rPilA) reacted specifically with convalescent and hyperimmune serum. Importantly, purified rPilA elicited a strong immune response and conferred robust protection against challenges with serovar 5 G. parasuis in mice. These results suggested that the PilA protein might help G. parasuis adhere to host cells by binding to FN and LN, and its immunogenicity establishes it as a promising, novel subunit vaccine candidate against infections with G. parasuis. IMPORTANCE G. parasuis is one of the most prevalent bacterial infections in swine production and can lead to huge economic losses around the world. A full understanding of colonization and immunity with G. parasuis infections will be essential in disease control. In this study, the PilA protein, which is a common virulence factor in other bacteria that mediates adherence to the host, was assessed. The results suggested that the PilA protein of G. parasuis can mediate adhesion to host cells through FN and LN, which provides a new idea for the study of the pathogenicity of G. parasuis . Furthermore, fimbriae usually have high immunogenicity. Immunogenicity and protective capacity results showed that the use of this recombinant PilA antigen might be a promising candidate vaccine antigen with which to prevent G. parasuis infections.

Glässer's disease is one of the main diseases affecting young piglets, particularly during the nursery phase, that can significantly impact pork production. Vaccination of sows has the potential to prevent Glaesserella parasuis infection during the first weeks of life that is to a substantial degree due to the transfer of maternal derived antibodies (MDA) in colostrum. In this study we compare the antibody response to two vaccines administered to pregnant sows. A subunit vaccine containing the mutant transferrin-binding protein, TbpB Y167A , and an autogenous vaccine formulated with the LM96/20 strain of G. parasuis (SV4) administered on days 65 and 86 of the gestational period were safe and induced high titers of antibodies in sows. The IgG peak was reached on day 100 of gestation, and the translocation of IgG to the mammary gland was confirmed in colostrum at the time of delivery. Piglets born from vaccinated sows maintained positive IgG titers against TbpB Y167A or G. parasuis SV4 for the duration of the experiment (35 days of life). Piglets born from sows vaccinated with the TbpB Y167A -based vaccine had a significantly (p = 0.001) lower load of G. parasuis in the respiratory tract compared to those born from sows vaccinated with the autogenous vaccine. Finally, we demonstrate that the LM96/20 (SV4) strain is highly virulent and a primary agent of Glässer's disease., Competing Interests: Conflicts of interest Dr. Schryvers is founder of Engineered Antigens Inc. a company that holds intellectual property related to the mutant Tbps. Dr. Frandoloso and Dr. Kreutz are partners of the biotech company, AFK Imunotech. None of the other authors has a financial or personal relationship with people or organizations that could inappropriately influence or bias the content of this study., (Copyright © 2022. Published by Elsevier B.V.)

Background: Glässer'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., Methods: In 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., Results: The 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 ., Conclusion: The overall results of the study suggest that the designed multi-epitope vaccine is a promising candidate for pan-prophylaxis against different strains of HPS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pang, Tu, Wang, Zhang, Ren, Yao, Luo and Yang.)

Glaesserella parasuis is a pathogen causing Glässer's disease characterized by fibrinous polyserositis, polyarthritis, and meningitis. Owing to the low cross-immunogenicity of different bacterial antigens in commercial vaccines, finding and identifying effective immunoprotective antigens will facilitate the development of novel subunit vaccines. In this study, LolA, identified by bioinformatics approaches, was cloned and successfully expressed as a recombinant protein in Escherichia coli, and its immunogenicity and protection were evaluated in a mouse model. The results showed that the recombinant protein LolA can stimulate mice to produce high levels of IgG antibodies and confer 50% protection against challenge with the highly virulent G. parasuis CY1201 strain (serotype 13). By testing the cytokine levels of interleukin 4 (IL-4), IL-10, and interferon-γ (IFN-γ), it was found that the recombinant protein LolA can induce both Th1 and Th2 immune responses in mice. These results suggest that the recombinant protein LolA has the potential to serve as an alternative antigen for a novel vaccine to prevent G. parasuis infection.

Glässer's disease in pigs is associated with infection by Glaesserella parasuis and is characterized by pneumonia-like symptoms, fibrinous polyserositis, polyarthritis, and meningitis. Macleaya cordata , a commonly used traditional Chinese medication, has been shown to have anti-inflammatory, antiviral, antioxidative, antimicrobial, insecticidal, and antitumor properties. However, the anti-inflammatory effects of M. cordata on G. parasuis stimulation are still poorly understood. This study explored the anti-inflammatory effects and mechanisms of M. cordata extract on G. parasuis -induced inflammatory responses, via the nuclear factor- kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, in porcine alveolar macrophages (PAMs). Porcine alveolar macrophages, when stimulated with G. parasuis , initiated transcription of interleukin (IL)-1α, IL-1β, IL-6, IL-8, and tumor necrosis factor alpha (TNF-α). Furthermore, p65, IκBα, p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) phosphorylation were upregulated via the NF-κB and MAPK signaling pathways. However, treatment with M. cordata extract inhibited transcription of IL-1α, IL-1β, IL-6, IL-8, and TNF-α and reduced p65, IκBα, p38, ERK, and JNK phosphorylation, by inhibiting activation of the NF-κB and MAPK signaling pathways in PAMs induced by G. parasuis . These findings reveal that M. cordata extract can reverse the inflammatory effect initiated by G. parasuis in vitro and that it possesses significant immunosuppression activity; thus, it may offer a novel strategy for controlling and treating G. parasuis infection., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

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., Competing Interests: Conflict of interest The authors declare that there is no conflict of interest., (Copyright © 2022. Published by Elsevier B.V.)

The YfeA gene, belonging to the well-conserved ABC (ATP-binding cassette) transport system Yfe , encodes the substrate-binding subunit of the iron, zinc, and manganese transport system in bacteria. As a potential vaccine candidate in Glaesserella parasuis , the functional mechanisms of YfeA in the infection process remain obscure. In this study, vaccination with YfeA effectively protected the C56BL6 mouse against the G. parasuis SC1401 challenge. Bioinformatics analysis suggests that YfeA is highly conserved in G. parasuis , and its metal-binding sites have been strictly conserved throughout evolution. Stimulation of RAW 264.7 macrophages with YfeA verified that toll-like receptors (TLR) 2 and 4 participated in the positive transcription and expression of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. The activation of TLR2 and TLR4 utilized the MyD88/MAL and TRIF/TRAM pairs to initiate TLRs signaling. Furthermore, YfeA was shown to stimulate nuclear translocation of NF-κB and activated diverse mitogen-activated protein (MAP) kinase signaling cascades, which are specific to the secretion of particular cytokine(s) in murine macrophages. Separate blocking TLR2, TLR4, MAPK, and RelA (p65) pathways significantly decreased YfeA-induced pro-inflammatory cytokine production. In addition, YfeA-stimulated RAW 264.7 produces the pro-inflammatory hallmark, reactive oxygen species (ROS). In conclusion, our findings indicate that YfeA is a novel pro-inflammatory mediator in G. parasuis and induces TLR2 and TLR4-dependent pro-inflammatory activity in RAW 264.7 macrophages through P38, JNK-MAPK, and NF-κB signaling pathways.

Glaesserella parasuis is well-known for causing Glässer's disease, which costs the worldwide swine industry millions of dollars each year. It has been reported the symptom of pleural thickening during Glässer's disease but this symptom has received little attention. And there is no research on the elements which promote pleural thickening. In this study, pleural thickening was discovered to be associated with increased collagen fibers and elastic fibers. Furthermore, collagen-I and elastin were found to be up-regulated and concentrated in the pleura at the mRNA and protein levels following infection. To summarize, our findings add to the theoretical understanding of Glässer's disease and provide strong support for further research into the pathogenic mechanism of Glaesserella parasuis and the program's target treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gong, Chen, He, Hua, Ma, Gao, Xu, Hu and Jin.)

Glaesserella parasuis (GPS), a causative agent of Glässer's disease, is thought to be the main fatal cause of peritonitis in swine, thus resulting in high mortality and morbidity and significant economic losses to the swine industry. However, the mechanisms of GPS infection-induced apoptosis and possible therapeutic pathway for GPS infection in peritonitis remain unclear. Baicalin has important biological functions during disease treatment, such as antiviral, bacterial inhibition, anti-apoptosis, and anti-inflammatory. However, whether baicalin has anti-apoptotic effects during the process of GPS infection in peritonitis is unclear. In the present study, the anti-apoptotic effect and mechanisms of baicalin in GPS infection-induced apoptosis were investigated in porcine peritoneal mesothelial cells (PPMC). The results showed that baicalin could inhibit the apoptosis rate occurrence of PPMC induced by GPS to various degrees and inhibit the expression of apoptosis-related genes and cleaved caspase-3. Meanwhile, baicalin significantly antagonized the expression of p-JNK, p-p38, and p-ERK induced by GPS in PPMC. These findings for the first time demonstrate that baicalin exerted the effect of antagonizing GPS induced apoptosis in PPMC by inhibiting the activation of the PKC-MAPK pathway and could be a therapeutic option in the management of GPS infection.

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.

Glaesserella parasuis is the etiological agent of Glässer's disease. Although present as a symbiotic bacterium in the respiratory tract of healthy pigs, G. parasuis invades piglets under stress conditions and causes severe systemic infection characterized by fibrinous polyserositis, polyarthritis, and meningitis, which caused high mortality in weaned and nursery herds. Further, the lack of cross-protection against the various serotypes of G. parasuis is a serious concern for the swine industry. Cytolethal distending toxin (CDT) is essential for the pathogenicity of G. parasuis and is a conserved virulence factor. CdtB is the active subunit of CDT, causing DNA double-strand breaks in eukaryotic cells, leading to irreversible cell cycle arrest and apoptosis. However, the immunogenicity and immunogenic domain of G. parasuis CdtB have not been investigated. In this study, monoclonal antibodies (mAbs) against G. parasuis CdtB were screened. One mAb, 4F10, was characterized and found to specifically recognize G. parasuis strains of all serotypes, including non-typeable strains, without showing any reactivity with other swine bacterial pathogens. Additionally, 4F10 exhibited neutralizing activity that restrained the cytotoxicity caused by CdtB. Moreover, the core unit of the epitope 84 GVGFPIDEYVWNLGTRSRPN 103 recognized by 4F10 was identified. The mAb-4F10 characterized herein provides a candidate for the further investigation of the pathogenic and immunogenic functions of CdtB in G. parasuis and could facilitate future serological diagnosis, prevention, and control of this disease., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

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 24 h /MIC for bacteriostasis, bactericidal action, and eradication were 5.10, 7.34, and 8.65 h and 5.91, 9.01, and 10.90 h in healthy and infected pigs, respectively. The optimal doses were 3.58-6.08 mg/kg in healthy pigs and 2.71-4.99 mg/kg in infected pigs from the point of view of preventing drug resistance., (© 2022 John Wiley & Sons Ltd.)

Haemophilus parasuis is a widespread bacterial pathogen causing acute systemic inflammation and leading to the sudden death of piglets. Resistin, a multifunctional peptide hormone previously demonstrated to influence the inflammation in porcine, was extremely increased in H. parasuis-infected tissues. However, the mechanism of resistin expression regulation in porcine, especially during pathogen infection, remains unclear. In the present study, we explored for the first time the transcription factor and signaling pathway mediating the expression of pig resistin during H. parasuis stimulation. We found that H. parasuis induced the expression of pig resistin in a time- and dose-dependent manner via the transcription factor Ets2 in porcine alveolar macrophages during H. parasuis stimulation. Moreover, the expression of Ets2 was mediated by the activation of the p38 MAPK pathway induced by H. parasuis, thus promoting resistin production. These results revealed a novel view of the molecular mechanism of pig resistin production during acute inflammation induced by pathogenic bacteria., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Co-infections of the porcine reproductive and respiratory syndrome virus (PRRSV) and the Haemophilus parasuis (HPS) are severe in Chinese pigs, but the immune response genes against co-infected with 2 pathogens in the lungs have not been reported., Objectives: To understand the effect of PRRSV and/or HPS infection on the genes expression associated with lung immune function., Methods: The expression of the immune-related genes was analyzed using RNA-sequencing and bioinformatics. Differentially expressed genes (DEGs) were detected and identified by quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry (IHC) and western blotting assays., Results: All experimental pigs showed clinical symptoms and lung lesions. RNA-seq analysis showed that 922 DEGs in co-challenged pigs were more than in the HPS group (709 DEGs) and the PRRSV group (676 DEGs). Eleven DEGs validated by qRT-PCR were consistent with the RNA sequencing results. Eleven common Kyoto Encyclopedia of Genes and Genomes pathways related to infection and immune were found in single-infected and co-challenged pigs, including autophagy, cytokine-cytokine receptor interaction, and antigen processing and presentation, involving different DEGs. A model of immune response to infection with PRRSV and HPS was predicted among the DEGs in the co-challenged pigs. Dual oxidase 1 ( DUOX1 ) and interleukin-21 ( IL21 ) were detected by IHC and western blot and showed significant differences between the co-challenged pigs and the controls., Conclusions: These findings elucidated the transcriptome changes in the lungs after PRRSV and/or HPS infections, providing ideas for further study to inhibit ROS production and promote pulmonary fibrosis caused by co-challenging with PRRSV and HPS., Competing Interests: The authors declare no conflicts of interest., (© 2022 The Korean Society of Veterinary Science.)

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.

Airway epithelial cells are the first line of defense against respiratory pathogens. Porcine bacterial pathogens, such as Bordetella bronchiseptica, Actinobacillus pleuropneumoniae, Glaesserella ( Haemophilus ) parasuis , and Pasteurella multocida , breach this barrier to lead to local or systematic infections. Here, we demonstrated that respiratory bacterial pathogen infection disrupted the airway epithelial intercellular junction protein, E-cadherin, thus contributing to impaired epithelial cell integrity. E-cadherin knocking-out in newborn pig tracheal cells via CRISPR/Cas9 editing technology confirmed that E-cadherin was sufficient to suppress the paracellular transmigration of these porcine respiratory bacterial pathogens, including G. parasuis, A. pleuropneumoniae, P. multocida , and B. bronchiseptica . The E-cadherin ectodomain cleavage by these pathogens was probably attributed to bacterial HtrA/DegQ protease, but not host HtrA1, MMP7 and ADAM10, and the prominent proteolytic activity was further confirmed by a serine-to-alanine substitution mutation in the active center of HtrA/DegQ protein. Moreover, deletion of the htrA gene in G. parasuis led to severe defects in E-cadherin ectodomain cleavage, cell adherence and paracellular transmigration in vitro , as well as bacterial breaking through the tracheal epithelial cells, systemic invasion and dissemination in vivo . This common pathogenic mechanism shared by other porcine respiratory bacterial pathogens explains how these bacterial pathogens destroy the airway epithelial cell barriers and proliferate in respiratory mucosal surface or other systemic tissues.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhang, Chu, Song, Yang, Bian, Li, Gou, Jiang, Cai and Li.)

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 (AUC 168h /MIC) positively correlated with the in vivo antibacterial effectiveness of tulathromycin ( R 2 = 0.9878 in serum and R 2 = 0.9911 in lung tissue). The computed doses to achieve a reduction of 2-log 10 CFU/lung from the ratios of AUC 72h /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 AUC 168h /MIC. In addition, using as objective a 2-log 10 reduction and an AUC 0-72h 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Guo, Gao, Wang, Lin, Fang and Zhao.)

Haemophilus (Glaesserella) parasuis is a commensal bacterium that causes Glässer's disease (GD) in swine. As a global transcriptional factor, CheY regulates the expression of hundreds of genes in H. parasuis. In this study, we measured changes in gene expression at the whole transcriptome level using RNAseq. We identified 2058 co-expressed genes, and found 624 differentially expressed genes (q < 0.05) in ΔcheY and SC1401. Several important GO annotations and signaling pathways were identified. RNA-seq results were assembled according to the reference genome, compared with the annotated gene model, and 12 new transcriptional regions were found. Finally, q-PCR results validated the RNA-seq results with 8 randomly selected genes. The present study indicated that CheY is mainly involved in the regulation of ABC transport, oxidative phosphorylation, and β-Lactam resistance. We draw the regulatory network of CheY, which offers greater insight into the regulatory mechanism of CheY in H.parasuis., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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.

Haemophilus parasuis is the main agent of Glässer'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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., Competing Interests: The authors declare no conflicts of interest., (© 2021 The Korean Society of Veterinary Science.)

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), HPS and PCV2 (p = .01, p < .05) and MHP and PRRSV (p = .01, p < .05). No significant correlation was observed between HPS and MHP (p = .067, 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., (© 2021 The Authors. Veterinary Medicine and Science Published by John Wiley & Sons Ltd.)

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., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Matiašková, Kavanová, Kulich, Gebauer, Nedbalcová, Kudláčková, Tesařík and Faldyna.)

Pathogens utilize various mechanisms to escape host immunological surveillance, break down different tissue barriers, and cause infection. Sialylation is an important surface modification of bacterial outer membrane components, especially the lipooligosaccharide of Gram-negative bacteria. It is widely involved in multiple microbe-host interactions, such as bacterial virulence regulation, host recognition, and immune evasion. There are some sialylation modifications on the lipooligosaccharide structure of Glaesserella parasuis ( G. parasuis ) virulent strains. However, the role of lipooligosaccharide sialylation modification in the process of G. parasuis infection and penetration of the porcine respiratory epithelial barrier is still unclear. In this study, we investigated the role and mechanism of lsgB -mediated lipooligosaccharide sialylation in G. parasuis invasion of the host respiratory epithelial barrier. Specifically, G. parasuis lsgB -mediated lipooligosaccharide sialylation and sialylated-lipooligosaccharide interacted with Siglec1 on porcine alveolar macrophages 3D4/21 and triggered the subsequent generation of TGFβ1 through Siglec1/Dap12/Syk/p38 signaling cascade. TGFβ1 decreased the tracheal epithelial tight junctions and the expression of extracellular adhesion molecule fibronectin, thus assisting G. parasuis invasion and entry to the respiratory epithelial barrier. Characterizing the potential effects and mechanisms of lipooligosaccharide sialylation-mediated TGFβ1 production would further expand our current knowledge on the pathogenesis of G. parasuis which will contribute to better prevention and control of G. parasuis infection in piglets.

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 MIC 50 and MIC 90 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 × 10 2 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.

Haemophilus parasuis induces severe acute systemic infection in pigs, characterized by fibrinous polyserositis, polyarthritis and meningitis. Our previous study demonstrated that H. parasuis induced the activation of p38 mitogen-activated protein kinase (MAPK) pathway, increasing the expression of proinflammatory genes and mediating H. parasuis-induced inflammation. Moreover, Wnt/β-catenin signaling activation induced by H. parasuis disrupts the adherens junction between epithelial cells and initiates the epithelial-mesenchymal transition (EMT). In the present study, p38 MAPK was found to be involved in the accumulation of nuclear location of β-catenin during H. parasuis infection in PK-15 and NPTr cells, via modulating the expression of dickkofp-1 (DKK-1), a negative regulator of Wnt/β-catenin signaling. We generated DKK-1 knockout cell lines by CRISPR/Cas9-mediated genome editing in PK-15 and NPTr cells, and found that knockout of DKK-1 led to the dysfunction of p38 MAPK in regulating Wnt/β-catenin signaling activity in H. parasuis-infected cells. Furthermore, p38 MAPK activity was independent of the activation of Wnt/β-catenin signaling during H. parasuis infection. This is the first study to explore the crosstalk between p38 MAPK and Wnt/β-catenin signaling during H. parasuis infection. It provides a more comprehensive view of intracellular signaling pathways during pathogenic bacteria-induced acute inflammation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 × 10 3  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., (© 2020 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Haemophilus parasuis (H. parasuis), the cause of the Glasser's disease, is a potentially pathogenic gram-negative organism that colonizes the upper respiratory tract of pigs. The extraction of Blumea balsamifera DC., as a traditional Chinese herb, has shown great bacteriostatic effect against several common bacteria. To study the antibacterial effect on H. parasuis in vitro, this study evaluated the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Blumea balsamifera DC. essential oil (BBO) as well as morphological changes in H. parasuis treated with it. Furthermore, changes in expression of total protein and key virulence factors were also assessed. Results showed that the MIC and MBC were 0.625 and 1.25 μg/mL, respectively. As the concentration of BBO increased, the growth curve inhibition became stronger. H. parasuis cells were damaged severely after treatment with BBO for 4 h, demonstrating plasmolysis and enlarged vacuoles, along with broken cell walls and membranes. Total protein and virulence factor expression in H. parasuis was significantly downregulated by BBO. Taken together, these results indicated a substantial antibacterial effect of BBO on H. parasuis.

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 10μg/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 (256μg/mL), tetracycline (128μg/mL), streptomycin (128μg/mL), gentamicin (128μg/mL), amoxicillin (128μg/mL), chloramphenicol (64μg/mL), penicillin (64μg/mL) and cefaclor (64μg/mL). Sequence analysis showed that numerous drug-resistance genes including tet(B), bla ROB-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-bla ROB-1 structure most likely had a direct plasmid origin. The tet(B) and bla ROB-1 were flanked on one or both by ISApl1 elements., Conclusions: The acquisition of bla ROB-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., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Haemophilus parasuis, a gram-negative bacterium as an early commensal colonizer in the upper respiratory tract of weaning pigs (Sus scrofa), is one of the most important bacterial pathogens affecting pig populations. It is the causative agent of Glässer's disease, causing systemic infection and polyserositis, meningitis, and arthritis. H. parasuis infection can result in high mortality and morbidity with, the significant economic losses for pig producers. To estimate the overall disease prevalence of H. parasuis in pigs from China, we performed a meta-analysis using five bibliographical databases: PubMed, ScienceDirect, CNKI, Wanfang, and VIP Chinese Journal Databases. A total of 41 articles published between 2005 and 2019, fulfilled the final inclusion criteria. The overall prevalence of H. parasuis in pigs in China was 27.8 % with the highest prevalence between 2011 and 2015 (41.0 %). In terms of pig age, the point estimate of H. parasuis prevalence was higher in suckling piglets (29.2 %) compared with that for other pig ages. The prevalence in the serum subgroup (29.8 %) was higher than that in the nasal swab subgroup (12.5 %). The results of the present meta-analysis showed that H. parasuis infection was common in pig populations in China; therefore, effective control measures are necessary to reduce this threat to pig populations., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Glässer's disease, caused by Haemophilus parasuis (H. parasuis), is associated with vascular damage and vascular inflammation in pigs. Therefore, early assessment and treatment are essential to control the inflammatory disorder. MicroRNAs have been shown to be involved in the vascular pathology. Baicalin has important pharmacological functions, including anti-inflammatory, antimicrobial and antioxidant effects. In this study, we investigated the changes of microRNAs in porcine aortic vascular endothelial cells (PAVECs) induced by H. parasuis and the effect of baicalin in this model by utilizing high-throughput sequencing. The results showed that 155 novel microRNAs and 76 differentially expressed microRNAs were identified in all samples. Subsequently, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the target genes of the differentially expressed microRNAs demonstrated that regulation of actin cytoskeleton, focal adhesion, ECM-receptor interaction, bacterial invasion of epithelial cells, and adherens junction were the most interesting pathways after PAVECs were infected with H. parasuis. In addition, when the PAVECs were pretreated with baicalin, mismatch repair, peroxisome, oxidative phosphorylation, DNA replication, and ABC transporters were the most predominant signaling pathways. STRING analysis showed that most of the target genes of the differentially expressed microRNAs were associated with each other. The expression levels of the differentially expressed microRNAs were negatively co-regulated with their target genes' mRNA following pretreatment with baicalin in the H. parasuis-induced PAVECs using co-expression networks analysis. This is the first report that microRNAs might have key roles in inflammatory damage of vascular tissue during H. parasuis infection. Baicalin regulated the microRNAs changes in the PAVECs following H. parasuis infection, which may represent useful novel targets to prevent or treat H. parasuis infection.

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