Haemophilus Parasuis Research Articles

Prevalence and time trend analysis of antimicrobial resistance in respiratory bacterial pathogens collected from diseased pigs in USA between 2006–2016

Swine respiratory disease complex (SRDC) causes massive economic losses to the swine industry and is a major animal welfare concern. Antimicrobials are mainstay in treatment and control of SRDC. However, there is a lack of data on the prevalence and trends in resistance to antimicrobials in bacterial pathogens associated with SRDC. The objective of this study was to estimate the prevalence and changes in resistance to 13 antimicrobials in swine bacterial pathogens (Streptococcus suis, Pasteurella multocida, Actinobacillus suis and Haemophilus parasuis) in the U.S.A using data collected at University of Minnesota Veterinary Diagnostic Laboratory between 2006 and 2016. For antimicrobials for which breakpoints were available, prevalence of resistance remained below 10% except for tetracycline in S. suis and P. multocida isolates, and these prevalence estimates remained consistently low over the years despite statistical significance (p < .05) in trend analysis. For antimicrobial-bacterial combinations without available breakpoints, the odds of isolates being resistant increased by >10% annually for 7 and 1 antimicrobials in H. parasuis and S. suis isolates respectively, and decreased >10% annually for 4 and 1 antimicrobials in A. suis and H. parasuis isolates, respectively, according to the ordinal regression models. Clinical implications of changes in AMR for A. suis and H. parasuis should be interpreted cautiously due to the lack of interpretive criteria and challenges in antimicrobial susceptibility tests in the case of H. parasuis. Future studies should focus on surveillance of antimicrobial resistance and establishment of standardized susceptibility testing methodologies and interpretive criteria for these animal pathogens of critical importance.

Efficacy of gamithromycin injection administered intramuscularly against bacterial swine respiratory disease

The aim of this study is to evaluate the safety and efficacy of gamithromycin (GAM) for the treatment of naturally occurring bacterial swine respiratory disease (SRD) administered IM. A total of 240 pigs (nine-weeks old) were selected from two sites in Heilongjiang Province of China. The pigs showed severe signs of respiratory disease. Among them, 120 pigs were randomly divided into 4 groups of low dose (3 mg/kg), middle dose (6 mg/kg), high dose (12 mg/kg) GAM IM injection and 2.5 mg/kg tulathromycin (TUL) IM injection (positive control group) for phase II clinical trial to screen effective therapeutic dose. The other 120 pigs were randomly divided into 2 groups of 6 mg/kg GAM IM injection and 2.5 mg/kg TUL IM injection (positive control group) for phase III clinical trial to further confirm the efficacy. Animals were clinically observed daily for 14 days after treatment initiation. The predominant pathogens present in pretreatment respiratory tract samples were Streptococcus suis (S. suis) and Actinobacillus pleuropneumoniae (A. pleuropneumoniae). Haemophilus parasuis (H. parasuis) and Pasteurella multocida (P. multocida) were also found in the respiratory tract. All isolates were subjected to in vitro sensitivity testing and the measured minimal inhibitory concentrations (MIC) of GAM were from 0.0625 μg/mL to 8 μg/mL. In all treatment groups, rectal temperature dropped and clinical index (mental status and respiratory symptom) significantly improved after treatment (P ≤ .05). As a result, 82.76% animals treated with the 6 mg/kg GAM injection were cured. This was significantly higher than that of 3 mg/kg GAM injection (P ≤ .05) and similar to that of 12 mg/kg GAM injection and 2.5 mg/kg TUL injection (P > .05) in phase II clinical trial. In phase III clinical trial, 80.70% of animals treated with the 6 mg/kg GAM injection were cured and the cure rate was similar to that of 2.5 mg/kg TUL injection (P > .05). In conclusion, we recommended a single dose (6 mg/kg) of GAM IM injection for the treatment of bacterial SRD.

Association between the Phenotypes and Genotypes of Antimicrobial Resistance in Haemophilus parasuis Isolates from Swine in Quang Binh and Thua Thien Hue Provinces, Vietnam

Haemophilus parasuis (H. parasuis) is one of the bacterial pathogens of great concern as it causes huge economic losses to the swine industry worldwide. One of the reasons why the control of H. parasuis has failed is the increase in antimicrobial resistance (AMR). The country of Vietnam has the second-largest pig production in Asia. However, there is still a lack of data about the AMR prevalence of H. parasuis in Vietnam. The purpose of this study is to investigate the prevalence of AMR and analyze the association between AMR and AMR genes (ARGs). The H. parasuis strains used in this research were isolated from swine in the Quang Binh and Thua Thien Hue Provinces, Central Vietnam, as reported in our previous study. All of the strains were tested for AMR against 25 antibacterial agents using the broth microdilution method and for the presence of ARGs using the polymerase chain reaction (PCR) method. The tested strains were shown to have a high frequency of resistance to trimethoprim/sulfamethoxazole (94.6%), followed by resistance to colistin, chloramphenicol, gentamicin, penicillin, lincomycin, and amoxicillin. The most prevalent ARGs in these strains were blaTEM-1 (94.6%), int (76.8%), gyrA (58.9%), and rmtD (50.0%). Cefuroxime, chloramphenicol, and tobramycin resistances were strongly correlated with the presence of the ARGs blaROB-1 (odds ratio (OR) = 26.3, 95% confidence interval (CI) 2.7–255.7, p = 0.002), catl (OR = 25.1, 95% CI 2.4–258.9, p = 0.004), and strB (OR = 23.5, 95% CI 2.6–212.6, p = 0.001), respectively. This study reveals for the first time the current situation of H. parasuis AMR in Central Vietnam, which is helpful for the clinical control of this disease, as well as for the development of policies and clinical practice guidelines to reduce AMR in swine production in Central Vietnam.

Phylogenomic analysis of Haemophilus parasuis and proposed reclassification to Glaesserella parasuis, gen. nov., comb. nov.

The Gram-negative bacterium Haemophilus parasuis is the etiologic agent of Glässer's disease in pigs, and causes significant economic losses to the swine industry. This bacterium has been classified as a member of the family Pasteurellaceae in the genus Haemophilus, but phylogenetic relatedness has not been adequately examined to support this genus classification. Phenotypically, all 38 strains of H. parasuis tested were positive for catalase activity, oxidase activity, V-factor requirement, and acid formation from maltose and d-galactose without gas. All strains were negative for X-factor requirement, formation of indole from tryptophan, urease, l-arabinose, and α-glucosidase activity. To determine whether H. parasuis belongs to one of the current Pasteurellaceae genera 40 H. parasuis genomes, plus those of representative Pasteurellaceae, were subjected to phylogenetic analysis of concatenated, multi-protein alignments. Sequence variation at 16S rRNA and rpoB loci allowed the 15 reference serovars of H. parasuis to be integrated into the whole-genome tree. The phylogenetic analysis showed H. parasuis to be a distinct and tight clade whose sister taxon is the genus Bibersteinia. Within H. parasuis two clades were identified with individual serovars distributed between the two. As a result, H. parasuis was confirmed as a member of the family Pasteurellaceae, but was distinct from other genera in this family. Therefore, we propose the name Glaesserella parasuis, gen. nov., comb. nov. for bacterial strains currently classified as H. parasuis. The reference strain of this species is ATCC 19417 (1374)T, NCTC 4557T, DSM 21448T, CCUG 3712T.

DETECTION OF PORCINE CIRCOVIRUS TYPE 3 IN RUSSIAN PIG HOLDINGS

Diseases associated with porcine circoviruses (mainly with porcine circovirus type 2) have various manifestations, are common in pigs in countries having well-developed pig industry and responsible for significant economic losses. Porcine circovirus type 3 (PCV-3) causing systemic inflammation of unknown etiology in animals was detected the USA in 2015. Later, data on PCV-3 detection in Asia, Europe and South America were published. Analysis of literature data on current epidemic situation on PCV-3 infection in foreign countries as well as the disease clinical manifestations and postmortem lesions are described. Results of molecular and genetic tests of biomaterials collected from pigs in 51 holdings located in 28 regions of the Russian Federation are presented. A total of 280 samples of biological materials of different types (organs, tissues, stillborn piglets) collected from domestic pigs with respiratory, reproductive and neurological disorders, dermatitis and from emaciated pigs were tested and PCV-3 genome was detected in 11 samples from 9 holdings located in 5 regions of the Russian Federation. Porcine circovirus type 3 was detected in lung, bronchial and mediastinal lymph node, spleen tissues from grower and fattening piglets, adult pigs and aborted fetuses. Samples that were positive for PCV-3 DNA when tested with molecular methods (PCR, real-time PCR) were tested for other pathogens. The following pathogens were also detected in 6 out of 11 samples (55%): Actinobacillus pleuropneumoniae, Mycoplasma hyorhinis, Streptococcus suis, Haemophilus parasuis, Mycoplasma hyopneumoniae and Pasteurella multocida. Porcine circovirus type 2 was detected in one sample. Presented test results are indicative of probable combined etiology of respiratory and reproductive disorders in tested pigs that results in various clinical manifestations. Grower and fattening piglets were found to be the most susceptible to PCV-3-associated disease. Further studies are required for identification of actual PCV-3 pathogenicity and its prevalence in the territory of the Russian Federation.

A crude capsular polysaccharide extract as a potential novel subunit vaccine with cross-protection against the most prevalent serovars of Glaesserella (Haemophilus) parasuis in the Czech Republic

Glaesserella (Haemophilus) parasuis is a part of the normal flora of the respiratory tract of pigs. However, under certain conditions it can also induce severe systemic disease with high morbidity and mortality leading to gross economic losses in the pig industry. The most prevalent serovars in pig herds in the Czech Republic are the virulent serovars 1, 4, 5 and 13. The currently available commercial vaccines are inactivated vaccines with certain limitations, such as no or poor cross-serovar protection. Therefore, the aim of the present study was to construct a subunit vaccine with a crude capsular polysaccharide extract (cCPS) isolated from G. parasuis CAPM 6475 (serovar 5) and evaluate its immunogenicity in a mouse model. Mice were immunised subcutaneously with two doses of the constructed vaccine in a 14-day interval and challenged intraperitoneally with various G. parasuis strains (serovars 1, 4, 5, 13) at 21 days after the second immunisation. The results of the ELISA test showed that the boost dose of the vaccine induced the production of IgG antibodies in high levels. On the basis of the death cases, the pathological findings and the bacterial isolation, the mice immunised with the cCPS were partially protected against the challenge with the homologous serovar 5 as well as with heterologous serovars 1, 4 and 13 of G. parasuis. The cross-reaction of the mixed serum from the immunised mice with the tested serovars was seen in the western-blotting also. Moreover, the most abundant protein found in the cCPS by mass spectrometry was catalase, a protein of molecular weight 55 kDa that may correspond to the strongest reaction seen in the western-blotting. Our findings indicated that the crude capsular polysaccharide extract may provide an effective immunogenicity in preventing a G. parasuis infection caused by the most prevalent serovars in the Czech Republic. However, the evaluation of the efficacy needs to be performed in pigs before any conclusions can be drawn.

Wnt/β-Catenin Signaling as a Molecular Target by Pathogenic Bacteria.

The Wnt/β-catenin signaling pathway is crucial to regulate cell proliferation and polarity, cell determination, and tissue homeostasis. The activation of Wnt/β-catenin signaling is based on the interaction between Wnt glycoproteins and seven transmembrane receptors—Frizzled (Fzd). This binding promotes recruitment of the scaffolding protein Disheveled (Dvl), which results in the phosphorylation of the co-receptor LRP5/6. The resultant molecular complex Wnt–Fzd–LRP5/6-Dvl forms a structural region for Axin interaction that disrupts Axin-mediated phosphorylation/degradation of the transcriptional co-activator β-catenin, thereby allowing it to stabilize and accumulate in the nucleus where it activates the expression of Wnt-dependent genes. Due to the prominent physiological function, the Wnt/β-catenin signaling must be strictly controlled because its dysregulation, which is caused by different stimuli, may lead to alterations in cell proliferation, apoptosis, and inflammation-associated cancer. The virulence factors from pathogenic bacteria such as Salmonella enterica sv Typhimurium, Helicobacter pylori, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Citrobacter rodentium, Clostridium difficile, Bacteroides fragilis, Escherichia coli, Haemophilus parasuis, Lawsonia intracellularis, Shigella dysenteriae, and Staphylococcus epidermidis employ a variety of molecular strategies to alter the appropriate functioning of diverse signaling pathways. Among these, Wnt/β-catenin has recently emerged as an important target of several virulence factors produced by bacteria. The mechanisms used by these factors to interfere with the activity of Wnt/β-catenin is diverse and include the repression of Wnt inhibitors' expression by the epigenetic modification of histones, blocking Wnt–Fzd ligand binding, activation or inhibition of β-catenin nuclear translocation, down- or up-regulation of Wnt family members, and inhibition of Axin-1 expression that promotes β-catenin activity. Such a variety of mechanisms illustrate an evolutionary co-adaptation of eukaryotic molecular signaling to a battery of soluble or structural components synthesized by pathogenic bacteria. This review gathers the recent efforts to elucidate the mechanistic details through which bacterial virulence factors modulate Wnt/β-catenin signaling and its physiological consequences concerning the inflammatory response and cancer.

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.

PK/PD modeling of Ceftiofur Sodium against Haemophilus parasuis infection in pigs

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

111 Common nutritional and infectious health challenges in nursery pigs

Poor starting nursery pigs are a common source of frustration for pork producers due to suboptimal lean tissue production and failure to thrive. This is generally a multifactorial issue with potential nutritional, infectious and management contributors. Commonly encountered respiratory and enteric pathogens include porcine reproductive and respiratory syndrome virus (PRRSV), influenza A virus (IAV), porcine enteric coronaviruses (TGEV/PEDV/PDCV), and group A, B, and C rotaviruses, as well as Salmonella typhimurium, enterotoxigenic Escherichia coli, Streptococcus suis, and Haemophilus parasuis. Infection with one or more of these agents can ultimately antagonize pig health and performance. However, while these specific pathogens may be causing an observed disease symptom, pigs may have been predisposed to infection due to various management, nutritional, and environmental risk factors. As many of these potential pathogens are endemic in production systems, it is important to remember that simply detecting a potential pathogen within a population is often not sufficient to assign cause for poor growth and production. To help fully interpret the impact of a detected agent, diagnostic efforts should focus on providing proof that the agent is actually causing disease. Molecular detection methods, such as PCR, are increasingly available for common pathogens and have high diagnostic sensitivity but lower diagnostic specificity. This paper will discuss the clinical signs and gross and microscopic lesions associated with common nursery pig pathogens, as well as proper sampling and diagnostic testing necessary to detect and confirm disease following infection with these agents.

Prevalence and antimicrobial susceptibilities of bacterial pathogens in Chinese pig farms from 2013 to 2017

Bacterial diseases of swine are a kind of multifactorial and uncontrollable diseases that commonly exist in pig farms all over the world and will lead to huge economic losses every year. In this study, a detailed and overall survey was carried out to better understand the prevalence and antimicrobial susceptibilities of bacterial diseases from 2013 to 2017 in China. A total of 19673 bacterial strains were isolated from 44175 samples collected from 9661 pig farms that distributed in 16 Chinese major pig breeding provinces. The results showed that the average isolation rates of Streptococcus suis (SS), Haemophilus parasuis (HPS), Escherichia coli (E. coli), Pasteurella multocida (Pm), Actinobacillus pleuropneumoniae (APP), Brodetella bronchiseptica (Bb), Salmonella enteria (SE), Erysipelothrix rhusiopathiae (E. rhusiopathiae) were 16.9%, 9.7%, 6.3%, 3.4%, 0.3%, 1.5%, 2.3% and 0.9%, respectively. The isolate rates of E. coli, APP and SE showed an increasing trend from 2013 to 2017. The seasonal prevalence characteristics of SS, HPS and Pm were obviously higher from April to August for first two bacteria and higher at February, March, April, and October for Pm. The dominant serotypes for SS, HPS were serotype 2 and serotype 5 (changed from serotype 4), respectively. The SS, HPS, and Pm showed very high antibiotic resistance rates to almost 8 common antibiotics (β-lactam, aminoglycoside, macrolides, lincomycin, tetracycline, quinolone, polymyxin, and sulfonamide) and an obvious increasing trend of antibiotic resistance rates from 2013 to 2017. In conclusion, the study provides detailed information on the prevalence and antimicrobial susceptibilities of different bacterial pathogens of swine from 2013 to 2017 in China. These data can provide a foundation for monitoring epidemiological patterns of bacterial diseases in the Chinese swine herds, as well as provide insight into potential antibiotic resistance profiles in these pathogens.

Baicalin modulates apoptosis via RAGE, MAPK, and AP-1 in vascular endothelial cells during Haemophilus parasuis invasion.

Glässer’s disease, caused by Haemophilus parasuis, is a chronic disease related to an inflammatory immune response. Baicalin exerts important biological functions. In this study, we explored the protective efficacy of treatment with baicalin and the potential mechanism of activation of the MAPK signaling pathway in porcine aortic vascular endothelial cells (PAVECs) induced by H. parasuis. H. parasuis stimulated expression of receptor for advanced glycation end products, induced a significant increase in the level of protein kinase-α and protein kinase-δ phosphorylation, and significantly up-regulated ERK, c-Jun N-terminal kinase, and p38 phosphorylation in PAVECs. H. parasuis also up-regulated the levels of apoptotic genes (Bax, C-myc, and Fasl) and the expression levels of c-Jun and c-Fos, and induced S-phase arrest in PAVECs. However, treatment with baicalin inhibited expression of RAGE, suppressed H. parasuis-induced protein kinase-α and protein kinase-δ phosphorylation, reduced ERK, c-Jun N-terminal kinase, and p38 phosphorylation, down-regulated apoptotic genes (Bax, C-myc, and Fasl), attenuated phospho-c-Jun production from the extracellular to the nuclei, and reversed S-phase arrest in PAVECs. In conclusion, baicalin treatment inhibited the MAPK signaling pathway, thereby achieving its anti-inflammatory responses, which provides a new strategy to control H. parasuis infection.

Application of PCR technique in diagnosis of four respiratory pathogenic bacteria in pigs at the slaughterhouse

The polymerase chain reaction (PCR) for Actinobacillus pleuropneumonia (App), Haemophilus parasuis (Hps), Pasteurella multocida (Pm) and Bordetella bronchiseptica (Bb) were performed in pure colonies isolated from 114 lung specimens with lesions collected from the Vissan slaughterhouse in Ho Chi Minh City from July 2018 to May 2019. The aim of the experiment was to identify the four respiratory pathogenic bacteria in pigs at slaughterhouse by using PCR technique. The criteria for evaluating the results included the proportion of positive samples with multiplex PCR and percentage of samples co-infected with 2, 3, and 4 bacteria. Among a total of 114 injured lung samples, 21% of the samples was positive to at least one of the four bacteria, 3 samples (2.63%) were positive for App, 2 samples (1.75%) were positive for Hps, 7 samples (6.14%) were for Pm, and 12 lungs (10.53%) were positive for Bb. One sample (0.88%) was found co-infected with Pm and Hps.

Construction of targeted and integrative promoter-reporter plasmids pDK-K and pDK-G to measure gene expression activity in Haemophilus parasuis

Haemophilus parasuis (H. parasuis) is rather difficult to manipulate genetically due to the diversity of restriction-modification systems and other mechanisms harbored by various isolates. This prevents exogenous plasmids from replicating in this species and hinders research efforts focused on transcriptional regulators in this bacterium. In this study, we generated a convenient promoter reporter system based on gene knock-in method using natural transformation in H. parasuis. Gene knock-in has proven useful as a powerful tool facilitating identification and studying the transcription activities of regulators under a variety of conditions that favor gene transcription or expression from an incorporated promoter. The vectors, pDK-K and pDK-G, carrying promoterless reporter lacZ gene and two homologous sequences flanking a knock-in site, may have some advantages over the extensively used plasmid-bearing reporter system in other bacteria in stability and ease of genetic manipulation in H. parasuis. The knock-in site was positioned at a site occupied by flanking genes that were both hypothetical and had the same transcription orientation, thus the expression of the reversely cloned promoter-lacZ fusion wouldn't be affected by the upstream promoter on the chromosome. The expression activity of lacZ gene under the transcriptional activation of a 300 bp promoter-proximal segment of cyaA, crp or comA genes in H. parasuis was separately validated using X-gal and o-nitrophenyl-β-d-galactoside(ONPG) as substrates. The derivatives harboring promoter-lacZ fusion segments showed significantly higher β-galactosidase activity levels than the promoterlessones both in TSB++ broth and on TSA++ plate as screened either by X-gal method or the standard Miller method. We also used pDK vector to further certify that the cyaA promoter is inducible and whose transcriptional levels were in correlation with the growth kinetics of the bacteria in TSB++. With this system, gene knock-in method based on natural transformation in H. parasuis proved to be useful in identifying transcriptional regulation of a certain promoter.

Genotypic analyses and virulence characterization of Glaesserella parasuis isolates from Taiwan.

BackgroundGlaesserella (Haemophilus) parasuis (G. parasuis) causes severe economic losses in the swine industry. Multiple G. parasuis strains can exist in single animals. Typing techniques are required for identifying G. parasuis isolates. Different strains within a serovar display varying virulence. Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) can assess the heterogeneity. The group 1 virulence-associated trimeric autotransporters (vtaA) gene is an indicator of virulence. The aim of this study was to characterize Taiwanese G. parasuis isolates via molecular serotyping, vtaA PCR and ERIC-PCR.MethodsOne hundred and forty-five strains were collected between November 2013 and March 2017 in Taiwan and further examined by molecular serotyping, vtaA PCR and ERIC-PCR.ResultsThe dendrogram revealed heterogeneous genetic diversity within many clusters. Partial correlation between the ERIC-PCR clusters of different strains, serovars and lesion patterns was observed. Twelve herds (8.3%) infected with more than one strain. Group 1 vtaA positive rate reached 98.6%.DiscussionThis study showed the high genetic diversity of G. parasuis in Taiwan by a high discriminatory capability of ERIC-PCR. Group 1 vtaA commonly exists in G. parasuis isolates and may play important roles in the pathogenesis of Taiwanese G. parasuis isolates.

Association between iscR-based phylogeny, serovars and potential virulence markers of Haemophilus parasuis.

Haemophilus parasuis is an economically important bacterial pathogen of swine. Extensive genetic and phenotypic heterogeneity among H. parasuis strains have been observed, which hinders the deciphering of the population structure and its association with clinical virulence. In this study, two highly divergent clades were defined according to iron–sulphur cluster regulator (iscR)-based phylogeny analysis of 148 isolates. Clear separation of serovars and potential virulence markers (PVMs) were observed between the two clades, which are indicative of independent evolution of the two lineages. Previously suggested virulence factors showed no correlation with clinical virulence, and were probably clade or serovar specific genes emerged during different stage of evolution. PVMs profiles varied widely among isolates in the same serovar. Higher strain diversity in respect of PVMs was found for isolates from multi-strain infected farms than those from single strain infected ones, which indicates that multi-strain infection in one farm may increase the frequency of gene transfer in H. parasuis. Systemic isolates were more frequently found in serovar 13 and serovar 12, while no correlation between clinical virulence and iscR-based phylogeny was observed. It shows that iscR is a reliable marker for studying population structure of H. parasuis, while other factors should be included to avoid the interference of gene exchange of iscR between isolates. The two lineages of H. parasuis may have undergone independent evolution, but show no difference in clinical virulence. Wide distribution of systemic isolates across the entire population poses new challenge for development of vaccine with better cross-protection. Our study provides new information for better deciphering the population structure of H. parasuis, which helps understanding the extreme diversity within this pathogenic bacterium.

Haemophilus parasuis infection in 3D4/21 cells induces autophagy through the AMPK pathway.

Haemophilus parasuis (H. parasuis) is a common commensal in the upper respiratory tract of pigs, but causes Glässer's disease in stress conditions. To date, many studies focused on the immune evasion and virulence of H.parasuis; very few have focused on the role autophagy played in H.parasuis infection, particularly in porcine alveolar macrophages (PAMs). In this study, a PAM cell line, 3D4/21 cells were used to study the role of autophagy in H.parasuis infection. 3D4/21 cells tandemly expressing GFP, mCherry, and LC3 were infected with H.parasuis serovar 5 (Hps5). Western blot analysis and confocal and transmission electron microscopy showed that H.parasuis infection effectively induces autophagy. Using Hps strains of varying virulence (Hps4, Hps5, and Hps7) and UV-inactivated Hps5, we demonstrated that autophagy is associated with the internalisation of living virulent strains into cells. In 3D4/21 cells pretreated with rapamycin and 3-MA then infected by Hps4, Hps5, and Hps7, we demonstrated that autophagy affects invasion of H.parasuis in cells. AMPK signal results showed that Hps5 infection can upregulate the phosphorylation level of AMPK, which is consistent with the autophagy development. 3D4/21 cells pretreated with AICAR or Compound C then infected by Hps5 revealed that the autophagy induced by Hps5 infection is associated with the AMPK pathway. Our study contributes to the theoretical basis for the study of H.parasuis pathogenesis and development of novel drugs target for prevention Glässer's disease.

Autophagy Is a Defense Mechanism Inhibiting Invasion and Inflammation During High-Virulent Haemophilus parasuis Infection in PK-15 Cells.

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

Antibacterial activity against porcine respiratory bacterial pathogens and in vitro biocompatibility of essential oils.

Bacterial respiratory infections affecting pigs such as pneumonia, pleuropneumonia, and pleurisy, are a major health concern in the swine industry and are associated with important economic losses. This study aimed to investigate the antibacterial activities of essential oils against major swine respiratory pathogens with a view to developing a potential alternative to antibiotics. Their synergistic interactions with the bacteriocin nisin was also examined. Lastly, we assessed the in vitro biocompatibility of the most efficient essential oils using a pig tracheal epithelial cell line. Of the nine essential oils tested, those from cinnamon, thyme, and winter savory were the most active against Streptococcus suis, Actinobacillus pleuropneumoniae, Actinobacillus suis, Bordetella bronchiseptica, Haemophilus parasuis, and Pasteurella multocida, with minimum inhibitory concentrations and minimum bactericidal concentrations ranging from 0.01 to 0.156% (v/v). The main component found in cinnamon, thyme, and winter savory oils were cinnamaldehyde, thymol, and carvacrol, respectively. Treating pre-formed S. suis and A. pleuropneumoniae biofilms with thyme or winter savory oils significantly decreased biofilm viability. We also observed a synergistic growth inhibition of S. suis with mixtures of nisin and essential oils from thyme and winter savory. Concentrations of nisin and cinnamon, thyme and winter savory essential oils that were effective against bacterial pathogens had no effect on the viability of pig tracheal epithelial cells. The present study brought evidence that essential oils are potential antimicrobial agents against bacteria associated with porcine respiratory infections.

Effects of Baicalin on piglet monocytes involving PKC\u2013MAPK signaling pathways induced by Haemophilus parasuis

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