Transcriptome analysis reveals a new virulence-associated trimeric autotransporter responsible for Glaesserella parasuis autoagglutination.

Capsular polysaccharide is an important virulence factor of Glaesserella parasuis. An acapsular mutant displays multiple phenotype variations, while the underlying mechanism for these variations is unknown. In this study, we created an acapsular mutant by deleting the wza gene in the capsule locus. We then used transcriptome analysis to compare the gene expression profiles of the wza deletion mutant with those of the parental strain to understand the possible reasons for the phenotypic differences. The mutant Δwza, which has a deleted wza gene, secreted less polysaccharide and lost its capsule structure. The Δwza exhibited increased autoagglutination, biofilm formation and adherence to eukaryotic cells, while the complementary strain C-Δwza partially restored the phenotype. Transcriptome analysis revealed several differentially expressed genes (DEGs) in Δwza, including up-regulated outer membrane proteins and proteins involved in peptidoglycan biosynthesis, suggesting that wza deletion affects the cell wall homeostasis of G. parasuis. Transcriptome analysis revealed the contribution of non-coding RNAs in the regulation of DEGs. Moreover, a new virulence-associated trimeric autotransporter, VtaA31 is upregulated in Δwza. It is responsible for enhanced autoagglutination but not for enhanced biofilm formation and adherence to eukaryotic cells in Δwza. In conclusion, these data indicate that wza affects the expression of multiple genes, especially those related to cell wall synthesis. Furthermore, they provide evidence that vtaA31 is involved in the autoagglutination of G. parasuis.

Glaesserella parasuis serotype 4 HPS4-YC disrupts the integrity of the swine tracheal epithelial barrier and facilitates bacterial translocation.

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.

The effects of baicalin on piglets challenged with Glaesserella parasuis.

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) 1ß, 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.

Haemophilus parasuis VtaA2 is involved in adhesion to extracellular proteins.

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.

Coinfection with Haemophilus parasuis serovar 4 increases the virulence of porcine circovirus type 2 in piglets.

BACKGROUND: Postweaning multisystemic wasting syndrome (PMWS) is an emerging disease in swine. Pigs with PMWS are often infected with a variety of other pathogens, including bacteria, viruses and mycoplasm, in addition to porcine circovirus type 2 (PCV2). PCV2 and Haemophilus parasuis serovar 4 (HPS4) coinfection remain epidemic in China. METHODS: Here we report construction of a three-week-old naturally farrowed, colostrum-deprived (NFCD) piglet's infection model and demonstrate that PCV2-infected piglets with the HPS4 coinfection increased the virulence of PCV2 and these pathogens interact acquired PMWS. RESULTS: All the single infected piglets were transiently bacteremic or viremic. All the PCV2/HPS4 coinfected piglets developed PMWS, characterized by dyspnea, anorexia, prostration and lose weight severely. Co-infection with PCV2 and HPS4 resulted in an increased amount of virus in serum and tissues, presented a slower generation and lower levels of antibodies against PCV2. Co-infection with PCV2 and HPS4 resulted in further reductions in total and differential peripheral blood leukocyte counts. Meantime, PCV2/ HPS4 coinfection potentiated the severity of lung and lymphoid lesions by PCV2-associated, increased the virulence of PCV2-antigen and enhanced the incidence of PMWS in piglets. CONCLUSION: Co-infection with PCV2 and HPS4 induce the exacerbation of system injuries and enhance the pathogenicity of PCV2 in piglets.

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

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

Haemophilus parasuis: infection, immunity and enrofloxacin.

Haemophilus parasuis is an early colonizer of the porcine upper respiratory tract and is the etiological agent of Glasser's disease. The factors responsible for H. parasuis colonization and systemic infection are not yet well understood, while prevention and control of Glasser's disease continues to be challenging. Recent studies on innate immunity to H. parasuis have demonstrated that porcine alveolar macrophages (PAMs) are able to differentially up-regulate several genes related to inflammation and phagocytosis, and several pro-inflammatory cytokines are produced by porcine cells upon exposure to H. parasuis. The susceptibility of H. parasuis strains to phagocytosis by PAMs and the bactericidal effect of complement are influenced by the virulent phenotype of the strains. While non-virulent strains are susceptible to phagocytosis and complement, virulent strains are resistant to both. However, in the presence of specific antibodies against H. parasuis, virulent strains become susceptible to phagocytosis. More information is still needed, though, in order to better understand the host immune responses to H. parasuis. Antimicrobials are commonly used in the swine industry to help treat and control Glasser's disease. Some of the common antimicrobials have been shown to reduce colonization by H. parasuis, which may have implications for disease dynamics, development of effective immune responses and immunomodulation. Here, we provide the current state of research on innate and adaptive immune responses to H. parasuis and discuss the potential effect of enrofloxacin on the development of a protective immune response against H. parasuis infection.

Deletion of the rfaE gene in Haemophilus parasuis SC096 strain attenuates serum resistance, adhesion and invasion.

In Haemophilus parasuis, the lipooligosaccharide (LOS) has been identified as an important virulence factor. The rfa gene cluster encodes enzymes for LOS core biosynthesis. In order to investigate the role of the rfaE gene, we generated an rfaE deficient mutant (ΔrfaE) of a H. parasuis SC096 by a natural transformation method. The purified preparation of LOS from the ΔrfaE mutant strain showed truncated LOS structure on silver-stained SDS-PAGE. Compared to the wild-type SC096 strain, the generation time of ΔrfaE mutant strain was significantly extended from 59 min to 69 min. The ΔrfaE mutant strain caused an approximately 30-fold reductions in survival rate in 50% sera and 36-fold reductions in survival rate in 90% sera, respectively (p < 0.001). In adhesion and invasion assays, the ΔrfaE mutant strain had 10-fold less efficient adherence and 12-fold reductions in invasion of the porcine umbilicus vein endothelial cells (PUVEC) and porcine kidney epithelial cells (PK-15), respectively (p < 0.001). However, the complemented strain could restore the above phenotypes. Hence, the above results suggested that the rfaE gene participated in the pathogenicity of H. parasuis SC096 strain.

Biofilm formation by virulent and non-virulent strains of Haemophilus parasuis.

Haemophilus parasuis is a commensal bacterium of the upper respiratory tract of healthy pigs. It is also the etiological agent of Glässer's disease, a systemic disease characterized by polyarthritis, fibrinous polyserositis and meningitis, which causes high morbidity and mortality in piglets. The aim of this study was to evaluate biofilm formation by well-characterized virulent and non-virulent strains of H. parasuis. We observed that non-virulent strains isolated from the nasal cavities of healthy pigs formed significantly (p < 0.05) more biofilms than virulent strains isolated from lesions of pigs with Glässer's disease. These differences were observed when biofilms were formed in microtiter plates under static conditions or formed in the presence of shear force in a drip-flow apparatus or a microfluidic system. Confocal laser scanning microscopy using different fluorescent probes on a representative subset of strains indicated that the biofilm matrix contains poly-N-acetylglucosamine, proteins and eDNA. The biofilm matrix was highly sensitive to degradation by proteinase K. Comparison of transcriptional profiles of biofilm and planktonic cells of the non-virulent H. parasuis F9 strain revealed a significant number of up-regulated membrane-related genes in biofilms, and genes previously identified in Actinobacillus pleuropneumoniae biofilms. Our data indicate that non-virulent strains of H. parasuis have the ability to form robust biofilms in contrast to virulent, systemic strains. Biofilm formation might therefore allow the non-virulent strains to colonize and persist in the upper respiratory tract of pigs. Conversely, the planktonic state of the virulent strains might allow them to disseminate within the host.

Characterization of an antimicrobial peptide produced by Bacillus subtilis subsp. spizezinii showing inhibitory activity towards Haemophilus parasuis.

Haemophilus parasuis is the pathogen that causes Glässer's disease, a major illness affecting young pigs. The aim of this work was to investigate the antagonistic activity of antimicrobial substances produced by Bacillus species against H. parasuis. Among the tested strains, only Bacillus subtilis ATCC 6633 inhibited H. parasuis growth. The antibacterial substance was purified by ammonium sulfate precipitation, gel filtration chromatography on Sephadex G-50 and ion-exchange chromatography on DEAE-cellulose. The purification was about 100-fold with a yield of 0.33 %. The purified substance was resistant up to 80 °C and pH ranging 3-7, but the substance lost its activity when it was treated with proteases. The peptide had a molecular mass of 1083 Da and its sequence was determined by MS as NRWCFAGDD, which showed no homology with other known antimicrobial peptides. The complete inhibition of H. parasuis growth was observed at 20 µg peptide ml(-1) after 20 min of exposure. The peptide obtained by chemical synthesis also showed antimicrobial activity on H. parasuis. The identification of antimicrobial substances that can be effective against H. parasuis is very relevant to combat this pathogen that causes important losses in swine production.

Differences in Haemophilus parasuis adherence to and invasion of AOC-45 porcine aorta endothelial cells.

BACKGROUND: The pathogenesis of Haemophilus parasuis depends on the bacterium's ability to interact with endothelial cells and invade adjacent tissues. In this study, we investigated the abilities of eight H. parasuis reference strains belonging to serovars 1, 2, 4, 5, 7, 9, 10 and 13 to adhere to and invade porcine aortic endothelial cells (AOC-45 cell line). RESULTS: The strains belonging to serovars 1, 2 and 5 were able to attach at high rates between 60 and 240 min of incubation, and serovars 4, 7 and 13 had moderate attachment rates; however, the strains belonging to serovars 9 and 10 had low adherence at all time points. Strong adherence was observed by scanning electron microscopy for the strains of serovars 5 and 4, which had high and moderate numbers, respectively, of H. parasuis cells attached to AOC-45 cells after 240 min of incubation. The highest invasiveness was reached at 180 min by the serovar 4 strain, followed by the serovar 5 strain at 240 min. The invasion results differed substantially depending on the strain. CONCLUSION: The reference strains of H. parasuis serovars 1, 2, 4 and 5 exhibited high adhesion and invasion levels to AOC-45 porcine aorta endothelial cells, and these findings could aid to better explain the pathogenesis of the disease caused by these serovars.

Transcription analysis on response of porcine alveolar macrophages to Haemophilus parasuis.

BACKGROUND: Haemophilus parasuis (H. parasuis) is the etiological agent of Glässer's disease in pigs. Currently, the molecular basis of this infection is largely unknown. The innate immune response is the first line of defense against the infectious disease. Systematical analysis on host innate immune response to the infection is important for understanding the pathogenesis of the infectious microorganisms. RESULTS: A total of 428 differentially expressed (DE) genes were identified in the porcine alveolar macrophages (PAMs) 6 days after H. parasuis infection. These genes were principally related to inflammatory response, immune response, microtubule polymerization, regulation of transcript and signal transduction. Through the pathway analysis, the significant pathways mainly concerned with cell adhesion molecules, cytokine-cytokine receptor interaction, complement and coagulation cascades, toll-like receptor signaling pathway, MAPK signaling pathway, suggesting that the host took different strategies to activate immune and inflammatory response upon H. parasuis infection. The global interactions network and two subnetworks of the proteins encoded by DE genes were analyzed by using STRING. Further immunostimulation analysis indicated that mRNA levels of S100 calcium-binding protein A4 (S100A4) and S100 calcium-binding protein A6 (S100A6) in porcine PK-15 cells increased within 48 h and were sustained after administration of lipopolysaccharide (LPS) and Poly (I:C) respectively. The s100a4 and s100a6 genes were found to be up-regulated significantly in lungs, spleen and lymph nodes in H. parasuis infected pigs. We firstly cloned and sequenced the porcine coronin1a gene. Phylogenetic analysis showed that poCORONIN 1A belonged to the group containing the Bos taurus sequence. Structural analysis indicated that the poCORONIN 1A contained putative domains of Trp-Asp (WD) repeats signature, Trp-Asp (WD) repeats profile and Trp-Asp (WD) repeats circular profile at the N-terminus. CONCLUSIONS: Our present study is the first one focusing on the response of porcine alveolar macrophages to H. parasuis. Our data demonstrate a series of genes are activated upon H. parasuis infection. The observed gene expression profile could help screening the potential host agents for reducing the prevalence of H. parasuis and further understanding the molecular pathogenesis associated with H. parasuis infection in pigs.

P38 MAPK pathway regulates the expression of resistin in porcine alveolar macrophages via Ets2 during Haemophilus parasuis stimulation.

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.

Biofilm characteristics and transcriptomic analysis of Haemophilus parasuis.

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.

Gene expression profiling in the lungs of pigs with different susceptibilities to Glässer's disease.

BACKGROUND: Haemophilus parasuis is the causative agent of Glässer's disease in pigs. Currently, little is known about the molecular mechanisms that contribute to disease susceptibility. This study used a porcine oligonucleotide microarray to identify genes that were differentially expressed (DE) in the lungs of colostrum-deprived animals previously characterized as being either 'Fully Resistant' (FR) or 'Susceptible' to infection by H. parasuis in a bacterial challenge experiment. RESULTS: Gene expression profiles of 'FR' and 'Susceptible' animals were obtained by the identification of genes that were differentially expressed between each of these groups and mock-inoculated 'Control' animals. At 24 hours post-inoculation, a total of 21 and 58 DE genes were identified in 'FR' and 'Susceptible' animals respectively. At 72 hours, the numbers of genes were 20 and 347 respectively. 'FR' animals at 24 hours exhibited an increased expression of genes encoding extracellular matrix and TGF-beta signalling components, possibly indicative of tissue repair following the successful early resolution of infection. The gene expression profile of 'FR' animals at 72 hours supported the hypothesis that higher levels of antibacterial activity were responsible for the 'FR' phenotype, possibly due to an increase in natural immunoglobulin A and decrease in signalling by the immunoregulatory transcription factor peroxisome proliferator-activated receptor gamma (PPAR-gamma). The expression profile of 'Susceptible' animals at both time-points was characterized by an imbalance in signalling between pro and anti-inflammatory cytokines and an increased expression of genes involved in biological processes associated with inflammation. These include the pro-inflammatory cytokine genes resistin (RETN) and interleukin 1-beta (IL1B). At 72 hours, a reduction in the expression of genes involved in antigen presentation by both MHC class I and II molecules was observed, which could have contributed to the inability of 'Susceptible' animals to clear infection. CONCLUSIONS: This study is the first to have identified discrete sets of DE genes in pigs of differing susceptibility to H. parasuis infection. Consequently, several candidate genes and pathways for disease resistance or susceptibility phenotypes have been identified. In addition, the findings have shed light on the molecular pathology associated with Glässer's disease.

Prevalence of Haemophilus parasuis"Glaesserella parasuis" in pigs in China: A systematic review and meta-analysis.

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.

COX-2 mediated crosstalk between Wnt/ß-catenin and the NF-κB signaling pathway during inflammatory responses induced by Haemophilus parasuis in PK-15 and NPTr cells.

Haemophilus parasuis infection causes typical acute systemic inflammation in pigs, is characterized by fibrinous polyserositis inflammation, and results in great economic losses to the swine industry worldwide. However, the molecular details of how the host modulates the acute inflammatory response induced by H. parasuis are largely unknown. In previous studies, we found that H. parasuis high-virulence strain SH0165 infection induced the activation of both Wnt/ß-catenin and NF-κB signaling in PK-15 and NPTr cells. In this study, we found that the activation of NF-κB, a central hub in inflammatory signaling, was impeded by the Wnt/ß-catenin pathway during H. parasuis infection. In contrast, blocking NF-κB activity had no effect on the Wnt/ß-catenin pathway during H. parasuis infection. Furthermore, we found that the inhibitory effect of ß-catenin on NF-κB activity was mediated by its target gene, pig cyclooxygenase-2 (COX-2). Therefore, we demonstrated that H. parasuis infection activates the canonical Wnt/ß-catenin signaling pathway, which leads to decreased NF-κB activity, reducing the acute inflammatory response in pigs. Additionally, the data provide a possible perspective for understanding the anti-inflammatory role of Wnt/ß-catenin in pigs during bacterial infection.

The Effect of Baicalin on the Expression Profiles of Long Non-Coding RNAs and mRNAs in Porcine Aortic Vascular Endothelial Cells Infected with Haemophilus parasuis.

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.

Effect of cAMP Receptor Protein Gene on Growth Characteristics and Stress Resistance of Haemophilus parasuis Serovar 5.

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

Identification of genes transcribed by Haemophilus parasuis in necrotic porcine lung through the selective capture of transcribed sequences (SCOTS).

Haemophilus parasuis is the aetiological agent of Glässer's disease, which has received more attention in the past decade due to the increasing economic losses in the pig industry worldwide. Little is known about the mechanisms by which H. parasuis survives in the host. In this study, selective capture of transcribed sequences (SCOTS) was used to identify H. parasuis genes upregulated in necrotic porcine lung 7 days post infection. Thirty-eight genes were identified that were upregulated during infection of the lung tissue of pigs, compared with growth in culture medium. In two examples chosen gene expression was not confined to the lungs, there being variation between tissues. The data support biofilm formation being an important mode of growth for colonization and/or persistence. Results from the in vitro studies suggest that, as for other pathogens, iron and oxygen restriction and heat stress are important environmental signals to regulate gene expression. This study has identified genes of H. parasuis that are upregulated during infection of porcine lung tissue as compared with in vitro growth conditions.