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.

Transcriptomic differences noted in Glaesserella parasuis between growth in broth and on agar.

Glaesserella parasuis is the cause of Glӓsser's disease in pigs and is a significant contributor to post-weaning mortality in the swine industry. Prevention of G. parasuis disease relies primarily on bacterin vaccines, which have shown good homologous protection and variable heterologous protection. Bacterin production involves large scale growth of the bacteria and proteins produced during the proliferation phase of production become important antigens that stimulate the immune response. In order to evaluate genes activated during G. parasuis growth on different media substrates, the transcriptome of broth and agar grown G. parasuis strain 29755 were sequenced and compared. The transcription of most purported virulence genes were comparable between broth and agar grown G. parasuis; however, four virulence-associated genes, including ompA and vapD, had elevated expression under agar growth, while six virulence-associate genes had elevated expression during broth growth, including several protease genes. Additionally, there were metabolic shifts toward increased protein and lipid production and increased cellular division in broth grown G. parasuis. The results contribute to the understanding of how growth substrate alters gene transcription and protein expression, which may impact vaccine efficacy if immunogens important to the protective immune response are not produced under specific in vitro conditions. While the results of this work are unable to fully elucidate which growth medium presents a transcriptome more representative of in vivo samples or best suited for bacterin production, it forms a foundation that can be used for future comparisons and provides a better understanding of the metabolic differences in broth and agar grown bacteria.

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.

The AI-2/luxS Quorum Sensing System Affects the Growth Characteristics, Biofilm Formation, and Virulence of Haemophilus parasuis.

Haemophilus parasuis (H. parasuis) is a kind of opportunistic pathogen of the upper respiratory tract of piglets. Under certain circumstances, virulent strains can breach the mucosal barrier and enter the bloodstream, causing severe Glässer's disease. Many virulence factors are found to be related to the pathogenicity of H. parasuis strain, but the pathogenic mechanism remains unclear. LuxS/AI-2, as a kind of very important quorum sensing system, affects the growth characteristics, biofilm formation, antibiotic production, virulence, and metabolism of different strains. In order to investigate the effect of luxS/AI-2 quorum sensing system on the virulence of H. parasuis, a deletion mutant strain (ΔluxS) and complemented strain (C-luxS) were constructed and characterized. The results showed that the luxS gene participated in regulating and controlling stress resistance, biofilm formation and virulence. Compared with wild-type strain, ΔluxS strain decreased the production of AI-2 molecules and the tolerance toward oxidative stress and heat shock, and it reduced the abilities of autoagglutination, hemagglutination, and adherence, whereas it increased the abilities to form biofilm in vitro. In vivo experiments showed that ΔluxS strain attenuated its virulence about 10-folds and significantly decreased its tissue burden of bacteria in mice, compared with the wild-type strain. Taken together, the luxS/AI-2 quorum sensing system in H. parasuis not only plays an important role in growth and biofilm formation, but also affects the pathogenicity of H. parasuis.

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.

Haemophilus parasuis Infection Disrupts Adherens Junctions and Initializes EMT Dependent on Canonical Wnt/ß-Catenin Signaling Pathway.

In this study, animal experimentation verified that the canonical Wnt/ß-catenin signaling pathway was activated under a reduced activity of p-ß-catenin (Ser33/37/Thr41) and an increased accumulation of ß-catenin in the lungs and kidneys of pigs infected with a highly virulent strain of H. parasuis. In PK-15 and NPTr cells, it was also confirmed that infection with a high-virulence strain of H. parasuis induced cytoplasmic accumulation and nuclear translocation of ß-catenin. H. parasuis infection caused a sharp degradation of E-cadherin and an increase of the epithelial cell monolayer permeability, as well as a broken interaction between ß-catenin and E-cadherin dependent on Wnt/ß-catenin signaling pathway. Moreover, Wnt/ß-catenin signaling pathway also contributed to the initiation of epithelial-mesenchymal transition (EMT) during high-virulence strain of H. parasuis infection with expression changes of epithelial/mesenchymal markers, increased migratory capabilities as well as the morphologically spindle-like switch in PK-15 and NPTr cells. Therefore, we originally speculated that H. parasuis infection activates the canonical Wnt/ß-catenin signaling pathway leading to a disruption of the epithelial barrier, altering cell structure and increasing cell migration, which results in severe acute systemic infection characterized by fibrinous polyserositis during H. parasuis infection.

Tea polyphenols suppress growth and virulence-related factors of Haemophilus parasuis.

The bacterium Haemophilus parasuis (H. parasuis) is the primary cause of Glässer's disease. Currently, there are no effective vaccines that can confer protection against all H. parasuis serovars. Therefore, the present study aimed to investigate the effect of tea polyphenols on growth, expression of virulence-related factors, and biofilm formation of H. parasuis, as well as to evaluate their protective effects against H. parasuis challenge. Our findings demonstrated that tea polyphenols can inhibit H. parasuis growth in a dose-dependent manner and attenuate the biofilm formation of H. parasuis. In addition, tea polyphenols exerted inhibitory effects on the expression of H. parasuis virulence-related factors. Moreover, tea polyphenols could confer protection against a lethal dose of H. parasuis and can reduce pathological tissue damage induced by H. parasuis. In summary, our findings demonstrated the promising use of tea polyphenols as a novel treatment for H. parasuis infection in pigs.

Baicalin modulates NF-κB and NLRP3 inflammasome signaling in porcine aortic vascular endothelial cells Infected by Haemophilus parasuis Causing Glässer's disease.

Haemophilus parasuis (H. parasuis) can cause vascular inflammatory injury, but the molecular basis of this effect remains unclear. In this study,we investigated the effect of the anti-inflammatory, anti-microbial and anti-oxidant agent, baicalin, on the nuclear factor (NF)-κB and NLRP3 inflammasome signaling pathway in pig primary aortic vascular endothelial cells. Activation of the NF-κB and NLRP3 inflammasome signaling pathway was induced in H. parasuis-infected cells. However, baicalin reduced the production of reactive oxygen species, apoptosis, and activation of the NF-κB and NLRP3 inflammasome signaling pathway in infected cells. These results revealed that baicalin can inhibit H. parasuis-induced inflammatory responses in porcine aortic vascular endothelial cells, and may thus offer a novel strategy for controlling and treating H. parasuis infection. Furthermore, the results suggest that piglet primary aortic vascular endothelial cells may provide an experimental model for future studies of H. parasuis infection.

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.

Deletion of the vacJ gene affects the biology and virulence in Haemophilus parasuis serovar 5.

Haemophilus parasuis is an important pathogen causing severe infections in pigs. However, the specific bacterial factors that participate in pathogenic process are poorly understood. VacJ protein is a recently discovered outer membrane lipoprotein that relates to virulence in several pathogens. To characterize the function of the vacJ gene in H. parasuis virulent strain HS49, a vacJ gene-deletion mutant ΔvacJ and its complemented strain were constructed. Our findings supported that VacJ is essential for maintenance of cellular integrity and stress tolerance of H. parasuis, by the demonstrations that the ΔvacJ mutant showed morphological change, increased NPN fluorescence and, and decreased resistance to SDS-EDTA, osmotic and oxidation pressure. The increased susceptibility to several antibiotics in the ΔvacJ mutant further suggested that the stability of the outer membrane was impaired as a result of the mutation in the vacJ gene. Compared to the wild-type strain, the ΔvacJ mutant strain caused a decreased survival ratio from the serum and complement killing, and exhibited a significant decrease ability to adhere to and invade PK-15 cell. In addition, the ΔvacJ mutant showed reduced biofilm formation compared to the wild-type strain. Furthermore, the ΔvacJ was attenuated in a murine (Balb/C) model of infection and its LD50 value was approximately fifteen-fold higher than that of the wild-type or complementation strain. The data obtained in this study indicate that vacJ plays an essential role in maintaining outer membrane integrity, stress tolerance, biofilm formation, serum resistance, and adherence to and invasion of host cells related to H. parasuis and further suggest a putative role of VacJ lipoprotein in virulence regulation.

The arcA gene contributes to the serum resistance and virulence of Haemophilus parasuis serovar 13 clinical strain EP3.

As a global transcriptional factor, ArcA regulates the expression of hundreds of genes involved in aerobic and anaerobic metabolism. Here we deleted arcA gene and investigated the biological characteristics of arcA deletion mutant (ΔarcA) in Haemophilus parasuis (H. parasuis) serovar 13 clinical strain EP3. Results indicated that deletion of arcA impaired growth of EP3 strain under anaerobic condition, and reduced virulence of EP3 strain in mice. Additionally, the ΔarcA strain showed greater sensitivity in porcine serum and produced less biofilm mass than the EP3 strain. Taken together, these findings suggested that the arcA gene may be involved in pathogenesis in Haemophilus parasuis.

Effect of cheY deletion on growth and colonization in a Haemophilus parasuis serovar 13 clinical strain EP3.

CheY, a response regulator of controlling the bacterial chemotactic swimming, can be modulated by either phosphorylation or acetylation to generate clockwise rotation of the flagella. Here, we researched the biological characteristics of cheY deletion mutant in Haemophilus parasuis, and found that the growth rate of this mutant was significantly slower compared with serovar 13 wild strain EP3. Additionally, the cheY mutant didn't show obvious sensitivity to porcine sera. The results of biofilm formation assay showed that H. parasuis cheY mutant produced less biofilm mass compared with wild strain. The H. parasuis cheY mutant reduced autoagglutination obviously. These findings were vital for revealing the function of cheY in growth, biofilm formation and autoagglutination. Thus, cheY plays a crucial role in growth and colonization in vivo of H. parasuis.

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.

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.

Gene expression profiling of Cecropin B-resistant Haemophilus parasuis.

Synthetically designed antimicrobial peptides (AMPs) present the potential of replacing antibiotics in the treatment of bacterial infections. However, microbial resistance to AMPs has been reported and little is known regarding the underlying mechanism of such resistance. The naturally occurring AMP cecropin B (CB) disrupts the anionic cell membranes of Gram-negative bacteria. In this study, CB resistance (CBR) was induced in Haemophilusparasuis SH0165 by exposing it to a series of CB concentrations. The CB-resistant H.parasuis strains CBR30 and CBR30-50 were obtained. The growth curves of SH0165 and CBR30 showed that CBR30 displayed lower growth rates than SH0165. The result of transmission electron microscopy showed cell membranes of the CB-resistant CBR30 and CBR30-50 were smoother than SH0165. Microarrays detected 257 upregulated and 254 downregulated genes covering 20 clusters of orthologous groups (COGs) of the CB-resistant CBR30 compared with SH0165 (>1.5-fold change, p < 0.05). Sixty genes were affected in CBR30-50 covering 18 COGs, with 28 upregulated and 32 downregulated genes. Under the COG function classification, the majority of affected genes in the CB-resistant CBR30 and CBR30-50 belong to the category of inorganic ion transport, amino acid transport, and metabolism. The microarray results were validated by real-time quantitative reverse transcription PCR. This study may provide useful guidance for understanding the molecular mechanism underlying H.parasuis resistance to CB.

Development of a neutralizing mouse-pig chimeric antibody with therapeutic potential against Haemophilus parasuis in Pichia pastoris.

Haemophilus parasuis is one of the most important bacterial diseases of pigs worldwide. The lack of a vaccine against a broad spectrum of strains and the limitation of antimicrobial susceptibility hamper the control of disease. In this study, we cloned the constant regions of gamma heavy chains and kappa light chain of pig lymphocytes in frame with the variable regions of heavy and light chains of mouse monoclonal antibody 1D8, which reacts with all 15 serotypes of H. parasuis and has neutralizing activity. The constructed mouse-pig chimeric antibody was expressed in Pichia pastoris. Results demonstrated that the expressed chimeric antibody inhibited the growth of H. parasuis in vitro. Furthermore, the experiments in mice showed that chimeric antibody increased survival rate of the mice compared with that of the control group (P < 0.05). Importantly, the chimeric antibody partially protected piglets against H. parasuis infection according to the clinical lesion scores and PCR results of H. parasuis in the tissues from piglets of the chimeric antibody-inoculated group and the PBS group. In summary, our results demonstrated that the mouse-pig chimeric antibody could be a therapeutic candidate to prevent the H. parasuis infection and control the prevalence of disease.

Transcriptional profiling of Haemophilus parasuis SH0165 response to tilmicosin.

The Haemophilus parasuis respiratory tract pathogen poses a severe threat to the swine industry despite available antimicrobial therapies. To gain a more detailed understanding of the molecular mechanisms underlying H. parasuis response to tilmicosin treatment, microarray technology was applied to analyze the variation in gene expression of isolated H. parasuis SH0165 treated in vitro with subinhibitory (0.25 µg/ml) and inhibitory (8 µg/ml) concentrations. Tilmicosin treatment induced differential expression of 405 genes, the encoded products of which are mainly involved in the heat shock response, protein synthesis, and intracellular transportation. The subinhibitory and inhibitory concentrations of tilmicosin induced distinctive gene expression profiles of shared and unique changes, respectively. These changes included 302 genes mainly involved in protein export and the phosphotransferase system to sustain cell growth, and 198 genes mainly related to RNA polymerase, recombination, and repair to inhibit cell growth. In silico analysis of functions related to the differentially expressed genes suggested that adaptation of H. parasuis SH0165 to tilmicosin involves modulation of protein synthesis and membrane transport. Collectively, the genes comprising each transcriptional profile of H. parasuis response to tilmicosin provide novel insights into the physiological functions of this economically significant bacterium and may represent targets of future molecular therapeutic strategies.

Comparative proteomic analysis of a Haemophilus parasuis SC096 mutant deficient in the outer membrane protein P5.

Outer membrane protein A (OmpA) is a major structural component of the outer membranes and functions as a multifaceted molecular with many diverse roles in Gram-negative bacteria. In Haemophilus parasuis, OmpA has been recognized and named as OmpP5 in genomic literature. In this study, to determine the precise functions of OmpP5, an ompP5 deficient mutant (ΔompP5) of a H. parasuis serovar 4 filed strain SC096 was constructed using a natural transformation method. Compared to the wild-type SC096 strain, the ΔompP5 mutant displayed a detectable delay in growth. However, the wild-type and mutant strains were indistinguishable with respect to the other phenotypes including resistance to killing by porcine and rabbit sera, adhesion to and invasion of porcine umbilicus veins endothelial cells (PUVEC) and porcine kidney epithelial cells (PK-15). To analyze the differences of proteome expression between wild-type and mutant strains, a 2-dimensional gel electrophoresis (2-DE)-based proteomics comparison was performed. There were 24 differentially expressed proteins which were mainly involved in carbohydrate, lipid, nucleotide and amino acid metabolism, or served as transcription and translation factors and chaperone proteins. Collectively, loss of OmpP5 expression in the H. parasuis SC096 strain resulted in global protein expression changes which might be responsible for novel phenotypes occurred in ΔompP5 mutant.

Non-viability of Haemophilus parasuis fur-defective mutants.

By complementation of an Escherichia coli fur mutant, the Haemophilus parasuis fur gene has been isolated from a genomic library of this organism. The H. parasuis fur gene is the distal one of a three-gene operon. Two genes placed upstream of the H. parasuis fur open-reading frame encode for a hypothetical protein and a flavodoxin, respectively. Attempts performed to isolate an H. parasuis fur-defective mutant either through manganese-resistance selection or exchange markers were unsuccessful. Likewise, anaerobic growth conditions do not enable the attainment of H. parasuis fur-defective mutants either. Nevertheless, H. parasuis clones carrying a knockout mutation in the chromosomal fur gene by insertion of a KmR cassette were obtained when a stable plasmid, containing an additional copy of the transcriptional unit to which the fur gene belongs, was present. Likewise, the presence of a plasmid in which the H. parasuis fur gene is under the control of the Escherichia coli tac promoter allows for the isolation of fur::Km mutants of this organism. Nonetheless, no fur-defective mutants may be isolated from H. parasuis cells harbouring a stable plasmid in which only the single fur gene is contained. These data clearly indicate that H. parasuis cell viability requires the presence of a wild-type fur gene.