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.

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.

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.

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.

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.

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.

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.

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.

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.

Basic Characterization of Natural Transformation in a Highly Transformable Haemophilus parasuis Strain SC1401.

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

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.

Either fadD1 or fadD2, Which Encode acyl-CoA Synthetase, Is Essential for the Survival of Haemophilus parasuis SC096.

In Haemophilus parasuis, the genes HAPS_0217 and HAPS_1695 are predicted to encode long-chain fatty acid-CoA ligases (FACSs). These proteins contain ATP/AMP signature motifs and FACS conserved motifs that are homologous to those in Escherichia coli FadD (EcFadD). In this study, we demonstrate that HAPS_0217 and HAPS_1695 can functionally replace EcFadD in the E. coli fadD mutant JW1794, and were thus designated fadD1 and fadD2, respectively. An evaluation of kinetic parameters indicated that FadD1 and FadD2 have a substrate preference for long-chain fatty acids. Moreover, FadD2 exhibited substrate inhibition in the presence of high concentrations of oleic acid. Single mutants of each of the fadD genes were easily constructed, whereas double mutants were not. These results were further confirmed using genomic site-directed mutagenesis, which supported the idea that H. parasuis requires either fadD1 or fadD2 for survival. The fadD1 mutant exhibited slower growth than the wild-type strain SC096, and its complementation resulted in a restored phenotype. The wild-type strain did not grow on chemically defined medium without the addition of oleic acid, indicating that lipids are a vital nutrient for this bacterium. Additionally, strains with a disrupted fadD1 gene also exhibited increased sensitivity to quinolone antibiotics, including levofloxacin, enrofloxacin, ciprofloxacin and nalidixic acid.

Haemophilus parasuis modulates cellular invasion via TGF-ß1 signaling.

TGF-ß1 plays an important role during the invasion of some bacteria into cells by regulating the expression of integrins, fibronectin, and other extracellular matrix proteins. We postulated that TGF-ß1 levels could affect the invasion of Haemophilus parasuis into PK-15 cells. After infection by H. parasuis, PK-15 cells had elevated levels of TGF-ß1 expression. Treatment of PK-15 cells with TGF-ß1 prior to infection significantly decreased invasion by H. parasuis. Both TGF-ß1 treatment and H. parasuis infection resulted in significant induction of fibronectin (Fn) and α5 integrin. Although pretreatment of PK-15 cells with siRNA fragments targeting Fn and α5 integrin resulted in enhanced H. parasuis invasion, H. parasuis attachment was elevated only on cells treated with the α5 integrin siRNA, there was no corresponding increase in attachment to cells treated with Fn siRNA. Our results firstly demonstrated that expression of TGF-ß1, Fn and α5 integrin inhibited invasion of H. parasuis in PK-15 cells.

The BALB/c mouse infection model for improving the Haemophilus parasuis serotyping scheme.

The use of BALB/c mouse as an alternative model to study Haemophilus parasuis (HPS) infections was evaluated, supplying the serotyping scheme by comparing the pathogenicity of different serovar HPS in pigs and mice challenge using statistical analysis. Results showed that the pathogenicity of different serovar HPS in mouse was consistent with in pigs, proving that this model is a viable alternative to pigs. It provides a convenient methodology for determining the virulence of HPS strains.

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.

Haemophilus parasuis infection activates chemokine RANTES in PK-15 cells.

RANTES is a member of the CC chemokine involved in inflammation and immune response during pathogen infection. In our previous study, Haemophilus parasuis (H. parasuis), which is responsible for the great economic losses in the pig industry worldwide, has been shown to enhance RANTES expression in PK-15 cells. However, the mechanisms behind this biological phenomenon have remained unclear. In this study, we showed that H. parasuis infection significantly upregulated RANTES gene transcription in a time- and dose-dependent manner. Promoter analysis by site-directed mutagenesis indicated that the nuclear factor NF-κB binding site was the most important cis-regulatory element controlling H. parasuis-induced RANTES transcription. Inhibition of NF-κB and JNK activity also significantly reduced H. parasuis-induced RANTES production. In addition, TLRs signaling pathway was found to be involved in H. parasuis induced-RANTES expression. These results represent an important molecular mechanism whereby H. parasuis induced RANTES in the inflammatory response.

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.

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 in Haemophilus parasuis: relationship with antibiotic resistance, serotype and genetic typing.

Biofilms are surface-associated microbial communities, which are encased in self-synthesized extracellular environment. Biofilm formation may trigger drug resistance and inflammation, resulting in persistent infections. Haemophilus parasuis is the etiological agent of a systemic disease, Glässer's disease, characterized by fibrinous polyserositis, arthritis and meningitis in pigs. The purpose of this study was to examine the correlation between biofilm and antibiotic resistance among the clinical isolates of H. parasuis. In the present study, we tested biofilm-forming ability of 110 H. parasuis isolates from various farms using polystyrene microtiter plate assays. Seventy-three isolates of H. parasuis (66.4%) showed biofilm formation and most of them performed weak biofilm-forming ability (38/73). All isolates were tested for antimicrobial susceptibility to 18 antimicrobial agents by the broth microdilution method. H. parasuis isolates showed very high resistance (>90%) to sulfanilamide, nalidixic acid, and trimethoprim. Resistance to eight antibiotics such as penicillin (41.1% vs 8.1%), ampicillin (31.5% vs 8.1%), amoxicillin (28.8% vs 5.4%), gentamicin (46.6% vs 24.3%), cefazolin (19.2% vs 2.7%), doxycycline (19.2% vs 8.1%), cefotaxime (11% vs 2.7%), and cefaclor (13.7% vs 5.4%) was comparatively higher among biofilm producers than non-biofilm producers. Pulsed-field gel electrophoresis (PFGE) analyses could distinguish various isolates. Our data indicated that H. parasuis field isolates were able to form biofilms in vitro. In addition, biofilm positive strains had positive correlation with resistance to ß-lactams antibiotics. Thus, biofilm formation may play important roles during H. parasuis infections.