Glaesserella parasuis serotype 5 breaches the porcine respiratory epithelial barrier by inducing autophagy and blocking the cell membrane Claudin-1 replenishment.

Glaesserella parasuis (G. parasuis), the primary pathogen of Glässer's disease, colonizes the upper respiratory tract and can break through the epithelial barrier of the respiratory tract, leading to lung infection. However, the underlying mechanisms for this adverse effect remain unclear. The G. parasuis serotype 5 SQ strain (HPS5-SQ) infection decreased the integrity of piglets' lung Occludin and Claudin-1. Autophagy regulates the function of the epithelial barrier and tight junction proteins (TJs) expression. We tested the hypothesis that HPS5-SQ breaking through the porcine respiratory epithelial barrier was linked to autophagy and Claudin-1 degradation. When HPS5-SQ infected swine tracheal epithelial cells (STEC), autophagosomes encapsulated, and autolysosomes degraded oxidatively stressed mitochondria covered with Claudin-1. Furthermore, we found that autophagosomes encapsulating mitochondria resulted in cell membrane Claudin-1 being unable to be replenished after degradation and damaged the respiratory tract epithelial barrier. In conclusion, G. parasuis serotype 5 breaks through the porcine respiratory epithelial barrier by inducing autophagy and interrupting cell membrane Claudin-1 replenishment, clarifying the mechanism of the G. parasuis infection and providing a new potential target for drug design and vaccine development.

TurboID Screening of the OmpP2 Protein Reveals Host Proteins Involved in Recognition and Phagocytosis of Glaesserella parasuis by iPAM Cells.

Glaesserella parasuis is a common bacterium in the porcine upper respiratory tract that causes severe Glasser's disease, which is characterized by polyarthritis, meningitis, and fibrinous polyserositis. TurboID is an enzyme that mediates the biotinylation of endogenous proteins that can fuse with proteins of interest to label protein interactors and local proteomes. To reveal the host proteins that interact with outer membrane protein P2 (OmpP2) by TurboID-mediated proximity labeling in immortalized porcine alveolar macrophage iPAM cells, 0.1 and 2.58 mg/mL His-tagged TurboID-OmpP2 and TurboID recombinant proteins were expressed and purified. By mass spectrometry, we identified 948 and 758 iPAM cell proteins that interacted with His-TurboID-OmpP2 and His-TurboID, respectively. After removal of background proteins through comparison with the TurboID-treated group, 240 unique interacting proteins were identified in the TurboID-OmpP2-treated group. Ultimately, only four membrane proteins were identified, CAV1, ARF6, PPP2R1A, and AP2M1, from these 240 host proteins. Our data indicated that CAV1, ARF6, and PPP2R1A could interact with OmpP2 of G. parasuis, as confirmed by coimmunoprecipitation assay. Finally, we found that CAV1, ARF6, and PPP2R1A were involved in the recognition and phagocytosis of G. parasuis serotype 5 by iPAM cells by using overexpression and RNA interference assays. This study provides first-hand information regarding the interaction of the iPAM cell proteomes with G. parasuis OmpP2 protein by using the TurboID proximity labeling system and identifies three novel host membrane proteins involved in the recognition and phagocytosis of G. parasuis by iPAM cells. These results provide new insight for a better understanding of Glasser's disease pathogenesis. IMPORTANCE G. parasuis can cause serious Glasser's disease, which is characterized by polyarthritis, meningitis, and fibrinous polyserositis in pigs. It can cause high morbidity and mortality in swine herds and major economic losses to the global pig industry. Understanding the mechanism of interactions between alveolar macrophages and pathogenic G. parasuis is essential for developing effective vaccines and targeted drugs against G. parasuis. To reveal the host proteins interacting with OmpP2 by TurboID-mediated proximity labeling in immortalized porcine alveolar macrophage (iPAM) cells, we identified 240 unique proteins from iPAM cells that could interact with G. parasuis OmpP2. Among them, only four membrane proteins, CAV1, ARF6, PPP2R1A, and AP2M1, were identified, and further study showed that CAV1, ARF6, and PPP2R1A are involved in the recognition and phagocytosis of G. parasuis serotype 5 by iPAM cells. This study provides new insight into proteomic interactions between hosts and pathogenic microorganisms.

Generation of markerless and multiple-gene knockout in Glaesserella parasuis based on natural transformation and Flp recombinase.

Glaesserella parasuis is an important bacterial pathogen that affects the swine industry worldwide. Research on the pathogenic mechanism and genetically engineered vaccine remains undeveloped because an effective markerless and multiple-gene knockout system is unavailable for G. parasuis yet. To establish a markerless knockout, deleted allelic genes with kanamycin resistance (KanR) cassettes were introduced into the genome of G. parasuis by using natural transformation with suicide plasmids. Then, the KanR cassette was excised with a thermosensitive plasmid pGF conferring a constitutive Flp expression. To realize the markerless and multiple-gene knockout, plasmid pGAF was constructed by placing the Flp gene under the control of an arabinose-inducible promoter. Firstly, pGAF was introduced into G. parasuis by electroporation, and the marked mutants were produced following natural transformation. Finally, the KanR cassette was excised from the genome by the inducible expression of Flp upon arabinose action. Based on the natural transformation and the inducible expression of Flp, the markerless single-gene knockout mutants of ΔhsdR, ΔneuA2, ΔespP2, Δapd, and ΔnanH were constructed. In addition, a five-gene knockout mutant of ΔhsdRΔneuA2ΔespP2ΔapdΔnanH was generated by successive natural transformation with five suicide plasmids. Taken together, a markerless and multiple-gene deletion system was established for G. parasuis in the present study for the first time. This system is simple, efficient, and easy to manipulate for G. parasuis; thus, our technique will substantially aid the understanding of the etiology, pathogenesis, and genetic engineering of G. parasuis and other bacteria that can be naturally transformed in laboratory conditions. KEY POINTS: • Flp recombinase excised the KanR gene flanked by FRT sites in Glaesserella parasuis. • The regulatory expression of Flp enabled a multiple-gene knockout forG. parasuis. • The technique will promote the understanding of Glässer's disease pathogens.

Impact of intracellular response regulator QseB in quorum sensing regulatory network in a clinical isolate SC1401 of Glaesserella parasuis.

Two component systems (TCS) mediate specific responses to different conditions and/or pressures. In the quorum sensing Glaesserella parasuis (QSE) BC TCS, qseB, as a response regulator, is closely related to the transcriptional regulation of multiple downstream genes. In this study, the effects of qseB gene deletion, which encodes the response regulator of population density sensing in G. parasuis, were studied through biological characteristics and metabolomic analysis. Based on previous research, we further explored the virulence of ΔqseB mutant strains through cell morphology, adhesion and invasion. The ΔqseB mutant and parent strains were sequenced by metabolome and combined with the previous transcriptome sequencing results for joint analysis. This study aims to clarify the regulatory effect of QseB on the virulence of G. parasuis and lay the foundation for revealing the pathogenic mechanism of G. parasuis. We detected 476 different metabolites, of which 30 metabolites (6.3%) had a significant difference in abundance between SC1401 and ΔqseB (p < 0.05). We conducted a comparative analysis of pathway enrichment on the transcriptome and metabolome, and found that the two omics participate in seven metabolic pathways together. The top 10 KEGG pathways with the largest number of genes and metabolites identified in this experiment are ABC transporters, Biosynthesis of secondary metabolites, Cysteine and methionine metabolism, Purine metabolism, Pyrimidine metabolism, Metabolic pathways, and Nicotinate and nicotinamide metabolism. Analysis of metabolome sequencing results showed that differential metabolites were also enriched in metabolic pathways, such as Purine metabolism, cGMP-PKG signaling pathway and cAMP signaling pathway, which were not found in transcriptome sequencing data. The internal coloration of the mutant strain ΔqseB was uneven, and the adhesion and invasion ability of PAM cell lines were significantly reduced. We speculate that QseB may affect the adhesion and invasion ability of Glaesserella parasuis by influencing substance transport and signal transduction.

Comparative transcriptome analysis reveals that deletion of CheY influences gene expressions of ABC transports and metabolism in Haemophilus parasuis.

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

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.

A novel nicotinamide adenine dinucleotide control strategy for increasing the cell density of Haemophilus parasuis.

Haemophilus parasuis is the causative agent of Glässer's disease and is a major source of economic losses in the swine industry each year. To enhance the production of an inactivated vaccine against H. parasuis, the availability of nicotinamide adenine dinucleotide (NAD) must be carefully controlled to ensure a sufficiently high cell density of H. parasuis. In the present study, the real-time viable cell density of H. parasuis was calculated based on the capacitance of the culture. By assessing the relationship between capacitance and viable cell density/NAD concentration, the NAD supply rate could be adjusted in real time to maintain the NAD concentration at a set value based on the linear relationship between capacitance and NAD consumption. The linear relationship between cell density and addition of NAD indicated that 7.138 × 109 NAD molecules were required to satisfy per cell growth. Five types of NAD supply strategy were used to maintain different NAD concentration for H. parasuis cultivation, and the results revealed that the highest viable cell density (8.57, OD600 ) and cell count (1.57 × 1010 CFU/mL) were obtained with strategy III (NAD concentration maintained at 30 mg/L), which were 1.46- and 1.45- times more, respectively, than cultures with using NAD supply strategy I (NAD concentration maintained at 10 mg/L). An extremely high cell density of H. parasuis was achieved using this NAD supply strategy, and the results demonstrated a convenient and reliable method for determining the real-time viable cell density relative to NAD concentration. Moreover, this method provides a theoretical foundation and an efficient approach for high cell density cultivation of other auxotroph bacteria.

Transcriptomics of Haemophilus (Glässerella) parasuis serovar 5 subjected to culture conditions partially mimetic to natural infection for the search of new vaccine antigens.

BACKGROUND: Haemophilus (Glässerella) parasuis is the etiological agent of Glässer's disease in pigs. Control of this disorder has been traditionally based on bacterins. The search for alternative vaccines has focused mainly on the study of outer membrane proteins. This study investigates the transcriptome of H. (G.) parasuis serovar 5 subjected to in vitro conditions mimicking to those existing during an infection (high temperature and iron-restriction), with the aim of detecting the overexpression of genes coding proteins exposed on bacterial surface, which could represent good targets as vaccine candidates. RESULTS: The transcriptomic approach identified 13 upregulated genes coding surface proteins: TbpA, TbpB, HxuA, HxuB, HxuC, FhuA, FimD, TolC, an autotransporter, a protein with immunoglobulin folding domains, another large protein with a tetratricopeptide repeat and two small proteins that did not contain any known domains. Of these, the first six genes coded proteins being related to iron extraction. CONCLUSION: Six of the proteins have already been tested as vaccine antigens in murine and/or porcine infection models and showed protection against H. (G.) parasuis. However, the remaining seven have not yet been tested and, consequently, they could become useful as putative antigens in the prevention of Glässer's disease. Anyway, the expression of this seven novel vaccine candidates should be shown in other serovars different from serovar 5.

OxyR of Haemophilus parasuis is a global transcriptional regulator important in oxidative stress resistance and growth.

Haemophilus parasuis is an opportunistic pathogen and the causative agent of Glässer's disease in swine. This disease has high morbidity and mortality rates in swine populations, and is responsible for major economic losses worldwide. Survival of H. parasuis within the host requires mechanisms for coping with oxidative stress conditions. In many bacteria, OxyR is known to mediate protection against oxidative stress; however, little is known about the role of OxyR in H. parasuis. In the current study, an oxyR mutant strain was constructed in H. parasuis strain SC1401 and designated H. parasuis SC1401∆oxyR. The oxyR mutant strain had a slower growth rate and impaired biofilm formation compared to the wild type strain. Complementation restored the growth-associated phenotypes to wild type levels. Oxidative stress susceptibility testing, using a range of concentrations of H2O2, indicated that H. parasuis SC1401∆oxyR was more sensitive to oxidative stress than the wild type strain. RNA sequencing transcriptome analysis comparing H. parasuis SC1401 with H. parasuis SC1401∆oxyR identified 466 differentially expressed genes. These genes were involved in a wide range of biological processes, including: oxidative stress, transcriptional regulation, and DNA replication, recombination, and repair. These findings provide a foundation for future research to examine the role of OxyR as a global transcriptional regulator and to better define its role in oxidative stress resistance in H. parasuis.

Haemophilus parasuis CpxRA two-component system confers bacterial tolerance to environmental stresses and macrolide resistance.

Haemophilus parasuis is an opportunistic pathogen localized in the upper respiratory tracts of pigs, its infection begins from bacterial survival under complex conditions, like hyperosmosis, oxidative stress, phagocytosis, and sometimes antibiotics as well. The two-component signal transduction (TCST) system serves as a common stimulus-response mechanism that allows microbes to sense and respond to diverse environmental conditions via a series of phosphorylation reactions. In this study, we investigated the role of TCST system CpxRA in H. parasuis in response to different environmental stimuli by constructing the ΔcpxA and ΔcpxR single deletion mutants as well as the ΔcpxRA double deletion mutant from H. parasuis serotype 4 isolate JS0135. We demonstrated that H. parasuis TCST system CpxRA confers bacterial tolerance to stresses and bactericidal antibiotics. The CpxR was found to play essential roles in mediating oxidative stress, osmotic stresses and alkaline pH stress tolerance, as well as macrolide resistance (i.e. erythromycin), but the CpxA deletion did not decrease bacterial resistance to abovementioned stresses. Moreover, we found via RT-qPCR approach that HAPS_RS00160 and HAPS_RS09425, both encoding multidrug efflux pumps, were significantly decreased in erythromycin challenged ΔcpxR and ΔcpxRA mutants compared with wild-type strain JS0135. These findings characterize the role of the TCST system CpxRA in H. parasuis conferring stress response tolerance and bactericidal resistance, which will deepen our understanding of the pathogenic mechanism in H. parasuis.

Comparative transcriptional profiling of tildipirosin-resistant and sensitive Haemophilus parasuis.

Numerous studies have been conducted to examine the molecular mechanism of Haemophilus parasuis resistance to antibiotic, but rarely to tildipirosin. In the current study, transcriptional profiling was applied to analyse the variation in gene expression of JS0135 and tildipirosin-resistant JS32. The growth curves showed that JS32 had a higher growth rate but fewer bacteria than JS0135. The cell membranes of JS32 and a resistant clinical isolate (HB32) were observed to be smoother than those of JS0135. From the comparative gene expression profile 349 up- and 113 downregulated genes were observed, covering 37 GO and 63 KEGG pathways which are involved in biological processes (11), cellular components (17), molecular function (9), cellular processes (1), environmental information processing (4), genetic information processing (9) and metabolism (49) affected in JS32. In addition, the relative overexpression of genes of the metabolism pathway (HAPS_RS09315, HAPS_RS09320), ribosomes (HAPS_RS07815) and ABC transporters (HAPS_RS10945) was detected, particularly the metabolism pathway, and verified with RT-qPCR. Collectively, the gene expression profile in connection with tildipirosin resistance factors revealed unique and highly resistant determinants of H. parasuis to macrolides that warrant further attention due to the significant threat of bacterial resistance.

lgtF effects of Haemophilus parasuis LOS induced inflammation through regulation of NF-κB and MAPKs signaling pathways.

The lgtF gene encodes a glucosyltransferase responsible for adding a glucose to the first sugar of heptose I in the synthesis of lipooligosaccharides (LOS). To study the function of lgtF, we constructed an lgtF mutant (ΔlgtF) from Haemophilus parasuis SC096 using a natural transformation system. A highly purified preparation of LOS from ΔlgtF (ΔlgtF-LOS) exhibited an obvious truncation in structure compared to the LOS of the wild-type SC096 strain (WT-LOS). The ΔlgtF-LOS also displayed a significantly reduced ability to induce inflammatory cytokine mRNA expression of tumor necrosis factor alpha (TNF-α), interleukin-1α (IL-1α), IL-1ß, IL-6 and IL-8 in porcine alveolar macrophages (PAMs) in comparison with the WT-LOS. Furthermore, we also found that ΔlgtF-LOS-treated cells had significantly decreased phospho-p65 and phospho-p38, and inhibited IκBα degradation. These findings suggested that the lgtF gene mediated LOS induction of pro-inflammatory cytokines in PAMs by regulating the NF-κB and MAPKs signaling pathways during H. parasuis infection.

The extended leader peptide of Haemophilus parasuis trimeric autotransporters conditions their protein expression in Escherichia coli.

Trimeric autotransporters are surface-exposed proteins of Gram-negative bacteria belonging to the type V secretion system. They are involved in virulence and are targets for vaccine and diagnostic tool development, so optimal systems for their expression and purification are required. In the present study, the impact of the extended leader peptide of the Haemophilus parasuis virulence-associated trimeric autotransporters (VtaA) in its production as recombinant proteins in Escherichia coli was evaluated. The 13 genes encoding the VtaA1 to VtaA13 passenger domains of the strain Nagasaki were cloned in the pASK-IBA33plus plasmid and expressed in E. coli. Recombinant protein production was higher for truncated forms in which the entire leader peptide was deleted, and the recombinant protein accumulated in the cytoplasm of the cells. The yield of protein production of the different VtaAs was size dependent, and reached maximal amount at 2-4 h post -induction. The optimization of these conditions allowed to scale-up the production to obtain enough recombinant protein to immunize large animals.

Identification of a surface epitope specific of virulent strains of Haemophilus parasuis.

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

Metatranscriptomics reveals metabolic adaptation and induction of virulence factors by Haemophilus parasuis during lung infection.

Haemophilus parasuis is a common inhabitant of the upper respiratory tract of pigs, and the causative agent of Glässer's disease. This disease is characterized by polyserositis and arthritis, produced by the severe inflammation caused by the systemic spread of the bacterium. After an initial colonization of the upper respiratory tract, H. parasuis enters the lung during the early stages of pig infection. In order to study gene expression at this location, we sequenced the ex vivo and in vivo H. parasuis Nagasaki transcriptome in the lung using a metatranscriptomic approach. Comparison of gene expression under these conditions with that found in conventional plate culture showed generally reduced expression of genes associated with anabolic and catabolic pathways, coupled with up-regulation of membrane-related genes involved in carbon acquisition, iron binding and pathogenesis. Some of the up-regulated membrane genes, including ABC transporters, virulence-associated autotransporters (vtaAs) and several hypothetical proteins, were only present in virulent H. parasuis strains, highlighting their significance as markers of disease potential. Finally, the analysis also revealed the presence of numerous antisense transcripts with possible roles in gene regulation. In summary, this data sheds some light on the scarcely studied in vivo transcriptome of H. parasuis, revealing nutritional virulence as an adaptive strategy for host survival, besides induction of classical virulence factors.

Sequence and structural diversity of transferrin receptors in Gram-negative porcine pathogens.

Actinobacillus pleuropneumoniae, Actinobacillus suis, and Haemophilus parasuis are bacterial pathogens from the upper respiratory tract that are responsible for a substantial burden of porcine disease. Although reduction of disease has been accomplished by intensive management practices, immunization remains an important strategy for disease prevention, particularly when intensive management practices are not feasible or suitable. An attractive target for vaccine development is the surface receptor involved in acquiring iron from host transferrin, since it is common to all three pathogenic species and has been shown to be essential for survival and disease causation. It has also recently been demonstrated that an engineered antigen derived from the lipoprotein component of the receptor, transferrin-binding protein B (TbpB), was more effective at preventing infection by H. parasuis than a commercial vaccine product. This study was initiated to explore the genetic and immunogenic diversity of the transferrin receptor system from these species. Nucleic acid sequences were obtained from a geographically and temporally diverse collection of isolates, consisting of 41 A. pleuropneumoniae strains, 30 H. parasuis strains, and 2 A. suis strains. Phylogenetic analyses demonstrated that the receptor protein sequences cluster independently of species, suggesting that there is genetic exchange between these species such that receptor-based vaccines should logically target all three species. To evaluate the cross-reactive response of TbpB-derived antigens, pigs were immunized with the intact TbpB, the TbpB N-lobe and the TbpB C-lobe from A. pleuropneumoniae strain H49 and the resulting sera were tested against a representative panel of TbpBs; demonstrating that the C-lobe induces a broadly cross-reactive response. Overall our results indicate that there is a common reservoir for transferrin receptor antigenic variation amongst these pathogens. While this could present a challenge to future vaccine development, our results suggest a rationally designed TbpB-based vaccine may provide protection against all three pathogens.

Deletion of HAPS_2096 Increases Sensitivity to Cecropin B in Haemophilus parasuis.

Cecropin B (CB) is a very effective natural antimicrobial peptide that has shown great potential for future antimicrobial drug development. HAPS_2096 is a Haemophilus parasuis gene that encodes the periplasmic substrate-binding protein of an ATP-binding cassette-type amino acid transporter. In this research, we constructed and verified an HAPS_2096 deletion mutant and a complementary HAPS_2096 mutant of H. parasuis JS0135. A bactericidal assay revealed that the HAPS_2096 deletion mutant was significantly more sensitive than the wild-type strain to 0.25-0.5 µg/ml CB. However, the gene complementation alleviated the CB sensitivity of the mutant. Immunoelectron microscopy observation following a 30-min treatment with a sublethal concentration of CB (0.25 µg/ml) revealed more extensive morphological damage in the mutant strain than in the wild-type strain. Hence, our results suggest that the HAPS_2096 gene contributes to H. parasuis resistance to CB.

Enhancement of Haemophilus parasuis serovar 5 yields by medium optimization.

UNLABELLED: The purpose of this study was to optimize the medium components for high productivity of Haemophilus parasuis serovar 5 through statistical approach. Plakett-Burman experimental design was initially applied to identify the factors that influenced the biomass of H. parasuis. Based on the response surface and canonical analyses, the optimum concentrations of the critical components were obtained as follows: 43·55 g l(-1) , yeast extract; 1·05 g l(-1) , sodium chloride; 11·63% (v/v), phosphate buffer; 10% (v/v), bovine serum; and 20 µg l(-1) , nicotinamide adenine dinucleotide. The number of viable cells of H. parasuis reached 4·7*10(9) CFU ml(-1) and the productivity was 4·7*10(9) CFU ml(-1) h(-1) after cultivation in the optimal medium in 3 l fermentor, increasing 2·5 times and 3·9 times more than that in tryptone soy broth medium, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report on statistical optimization of medium components for the fermentation of Haemophilus parasuis serovar 5. The improved medium was highly efficient, less expensive (its cost was $1·16 l(-1) , while that of tryptone soy broth was $4·00 l(-1) ) and has been used for large-scale production in Wuhan Keqian Animal Biology Product Co. Ltd, China, and it will improve the industrialization of H. parasuis worldwide.

Role of acrAB in antibiotic resistance of Haemophilus parasuis serovar 4.

Haemophilus parasuis infection is responsible for important economic losses to the pig industry. The increasing emergence of resistance to multiple antibiotics is of concern and to study the role of the acrRAB operon in H. parasuis drug resistance, acrB or acrR mutants were generated from H. parasuis serovar 4 clinical strains. The susceptibilities of the clinical strains and their acrB/acrR mutants to a number of antibiotics were determined. The acrB mutants were more susceptible to novobiocin, erythromycin, clarithromycin, and azithromycin. In the acrR mutant of H. parasuis, acrB was up-regulated, as determined by quantitative reverse transcriptase polymerase chain reaction. The results of this study indicated that the efflux pump AcrB may play a role in multidrug resistance of H. parasuis.