Adjuvant Efficacy of the ECMS-Oil on Immune Responses against Bordetella bronchiseptica in Mice through the TLR2/MyD88/NF-κB Pathway.

Bordetella infection can be efficiently prevented through vaccination. The current study investigated the effects of an extract of Cochinchina momordica seed (ECMS) combined with oil on the immune responses to the inactivated Bordetella vaccine in mice. Serum IgG and IgG1 level was significantly increased in ECMS-oil group compared to any other group (P < 0.05) 2 weeks after immunization, while groups ECMS200 µg/400 µg-oil had a markedly higher level of serum IgG2b and IgG3 than any other groups (P < 0.05). Moreover, lipopolysaccharide/ConA-stimulated proliferation of splenocytes was significantly enhanced in ECMS 400 µg-oil immunized mice in comparison with mice in any other group (P < 0.05). RT-PCR assay revealed that while ECMS800 µg-oil group had significantly higher levels of serum IL-4, IL-10, Toll-like receptor (TLR)2, and IL-1 beta than any other group (P < 0.05), the levels of serum IL-2, IL-4, and IL-10 were markedly increased in ECMS 400 µg-oil group as compared to any other groups (P < 0.05). Blood analysis showed that ECMS800 µg-oil and oil groups had a significantly higher number of immunocytes than any other groups (P < 0.05). There were significant differences in the number of IgG+, IgG2b+, and IgA+ cells in the lung between ECMS800 µg-oil group and any other groups (P < 0.05). Western blot analysis demonstrated that stimulation with ECMS 25 µg/mL or 50 ng/mL led to a significant increase in the expression of TLR2, MyD88, and NF-κB in Raw264.7 cells (P < 0.05). Compared with any other group, the expression of MyD88 was markedly increased in the cells stimulated with ECMS 50 ng/mL, as indicated by the RT-PCR analysis (P < 0.05). Overall, we observed that ECMS-oil efficiently enhanced the humoral or cellular immune responses against Bordetella and suggested that the mechanism of adjuvant activity of ECMS-oil might involve TLR2/MyD88/NF-κB signaling pathway.

Using least angular regression to model the antibacterial potential of metronidazole complexes.

Imidazole has anti-inflammatory, antituberculotic, antimicrobial, antimycotic, antiviral, and antitumor properties in the human body, to name a few. Metronidazole [1-(2-Hydroxyethyl)-2-methyl-5-nitroimidazole] is a widely used antiprotozoan and antibacterial medication. Using fourier transform infrared spectroscopy, the current study models the antibacterial activity of already synthesised Metronidazole (MTZ) complexes ([Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text] and [Formula: see text]) against E. coli, B. bronceptica, S. epidermidis, B. pumilus and S. aureus. To characterise the Metronidazole complexes for antibacterial activity against 05 microbes, the least angular regression and least absolute shrinkage selection operators were used. Asymmetric Least Squares was used to correct the spectrum baseline. Least angular regression outperforms cross-validated root mean square error in the fitted models. Using Least angular regression, influential wavelengths that explain the variation in antibacterial activity of Metronidazole complexes were identified and mapped against functional groups.

Green Synthesis of MnO Nanoparticles Using Abutilon indicum Leaf Extract for Biological, Photocatalytic, and Adsorption Activities.

We report the synthesis of MnO nanoparticles (AI-MnO NAPs) using biological molecules of Abutilon indicum leaf extract. Further, they were evaluated for antibacterial and cytotoxicity activity against different pathogenic microbes (Escherichia coli, Bordetella bronchiseptica, Staphylococcus aureus, and Bacillus subtilis) and HeLa cancerous cells. Synthesized NAPs were also investigated for photocatalytic dye degradation potential against methylene blue (MB), and adsorption activity against Cr(VI) was also determined. Results from Scanning electron microscope (SEM), X-ray powder diffraction (XRD), Energy-dispersive X-ray (EDX), and Fourier-transform infrared spectroscopy (FTIR) confirmed the successful synthesis of NAPs with spherical morphology and crystalline nature. Biological activity results demonstrated that synthesized AI-MnO NAPs exhibited significant antibacterial and cytotoxicity propensities against pathogenic microbes and cancerous cells, respectively, compared with plant extract. Moreover, synthesized AI-MnO NAPs demonstrated the comparable biological activities results to standard drugs. These excellent biological activities results are attributed to the existence of the plant's biological molecules on their surfaces and small particle size (synergetic effect). Synthesized NAPs displayed better MB-photocatalyzing properties under sunlight than an ultraviolet lamp. The Cr(VI) adsorption result showed that synthesized NAPs efficiently adsorbed more Cr(VI) at higher acidic pH than at basic pH. Hence, the current findings suggest that Abutilon indicum is a valuable source for tailoring the potential of NAPs toward various enhanced biological, photocatalytic, and adsorption activities. Consequently, the plant's biological molecule-mediated synthesized AI-MnO NAPs could be excellent contenders for future therapeutic applications.

Bordetella bronchiseptica pneumonia in a patient with lung cancer; a case report of a rare infection.

BACKGROUND: Bordetella bronchiseptica (B.bronchiseptica) is a frequent cause of respiratory infections in animals but rarely causes serious infection in humans. We present a rare case of B. bronchiseptica pneumonia in a patient with lung cancer. CASE PRESENTATION: A 52-year-old white male with non small cell lung cancer developed fever during treatment with nivolumab. A persistent productive cough and a deterioration in his clinical condition led to his hospitalization for evaluation. Bronchoscopy was performed and a diagnosis of B. bronchiseptica pneumonia was made. The infection was successfully managed by antiobiotic therapy. CONCLUSIONS: B. bronchiseptica is a pathogen that can cause serious infection in humans, especially in immunocompromised or immunoincompetent individuals. In our patient it showed unusual resistance to cephalosporins and poor sensitivity to amikacin. To our knowledge this is the first case of such an infection in a lung cancer patient undergoing treatment with nivolumab. When B. bronchiseptica is identified, the possibility of a nosocomial transmission must be considered.

Acid tolerance response of Bordetella bronchiseptica in avirulent phase.

Bordetella bronchiseptica is a Gram-negative bacterium responsible for respiratory diseases in many mammalian hosts, including humans. This pathogen has been shown as able to persist inside the host cells, even in the phagosomes that are acidified to pH 4.5-5.0 after bacterial infection. Here we evaluated the resistance of B. bronchiseptica to survive under acidic conditions. In particular we analyzed the bacterial capacity to develop the mechanism known as acid tolerance response (ATR). Our studies were mainly focused on the avirulent phase of the bacteria since this phenotypic phase was reported to be more resistant to environmental stress conditions than the virulent phase. Results from B. bronchiseptica in virulent phase were also included for comparison purposes. In fact, for B. bronchiseptica 9.73 bacteria in virulent phase we observed that the viability of bacteria does not decrease significantly when grown at pH as low as 4.5, but it is affected when the pH of the medium was equal to or less than 4.0. After acid-adaptation at pH 5.5 for several hours, the survival rate of B. bronchiseptica 9.73 at lethal pH 4.0 for 6h was increased. Interestingly, the avirulent phase mediated by the two-component BvgAS system conferred further resistance to lethal acid challenge and a marked increase in the magnitude of the expressed ATR. The ATR for this avirulent phase seems to be associated with changes in LPS and surface protein profiles. 2D-gel electrophoresis revealed at least 25 polypeptides differentially expressed, 17 of which were only expressed or over-expressed under acid conditions. Using MALDI-TOF mass spectrometry, 10 of these differentially expressed polypeptides were identified.

Bacteriemia recurrente por Bordetella bronchiseptica en un paciente con trasplante de medula ósea / Bordetella bronchiseptica recurrent bacteraemia in a patient with bone marrow transplantation

Se reporta un caso de bacteriemia recurrente por Bordetella bronchiseptica en un paciente inmunocomprometido con antecedentes de trasplante alogénico de medula ósea por síndrome mielodisplásico, quien ingresó al hospital por síndrome febril. Bordetella bronchiseptica es un agente patógeno veterinario poco común en humanos que afecta principalmente a pacientes inmunocomprometidos y es causa poco frecuente de bacteriemia.

We report a case of recurrent bacteraemia caused by Bordetella bronchiseptica in an immunocompromised patient with a history of allogenic bone marrow transplantation for myelodysplastic syndrome, who was admitted to hospital with febrile syndrome. Bordetella bronchiseptica is an uncommon human pathogen which mainly affects immunocompromised patients, being a rare cause of bacteraemia.

Antimicrobial, antitumor and brine shrimp lethality assay of Ranunculus arvensis L. extracts.

To investigate the antitumor activity, brine shrimp lethality assay, antibacterial and antifungal activity of Methanol Extract (ME), Water Extract (WE), Acetone Extract (AE), Chloroform Extract (CE), Methanol-Water Extract (MWE), Methanol-Acetone Extract (MAE), Methanol-Chloroform Extract (MCE) of Ranunculus arvensis (L.). Antitumor activity was evaluated with Agrobacterium tumefaciens (At10) induced potato disc assay. Cytotoxicity was evaluated with brine shrimp lethality assay. Antibacterial activity was evaluated with six bacterial strains including Escherichia coli, Enterobacter aerogenes, Bordetella bronchiseptica, Klebsiella pneumoniae, Micrococcus luteus and Streptococcus anginosus and antifungal screening was done against five fungal strains including Aspergillus niger, A. flavus, A. fumigates, Fusarium solani and Mucor species by using disc diffusion method. Best antitumor activity was obtained with ME and WE, having highest IC50 values 20.27 ± 1.62 and 93.01 ± 1.33µg/disc. Brine shrimp lethality assay showed LC50 values of AE, MAE and ME were obtained as 384.66 ± 9.42µg/ml, 724.11 ± 8.01µg/ml and 978.7 ±8.01 µg/ml respectively. WE of R. arvensis revealed weak antimicrobial result against the tested microorganisms. On the other hand, the antifungal activity of the plant extracts was found to be insignificant. These findings demonstrate that extracts of R. arvensis possesses significant antitumor activity. Further extensive study is necessary to assess the therapeutic potential of the plant.

[Bordetella bronchiseptica recurrent bacteraemia in a patient with bone marrow transplantation]. / Bacteriemia recurrente por Bordetella bronchiseptica en un paciente con trasplante de medula ósea.

We report a case of recurrent bacteraemia caused by Bordetella bronchiseptica in an immunocompromised patient with a history of allogenic bone marrow transplantation for myelodysplastic syndrome, who was admitted to hospital with febrile syndrome. Bordetella bronchiseptica is an uncommon human pathogen which mainly affects immunocompromised patients, being a rare cause of bacteraemia.

Immunoprophylactic effects of shiitake mushroom (Lentinula edodes) against Bordetella bronchiseptica in mice.

Antimicrobials are used as feed additives to improve growth performance and to prevent subclinical disease challenge in industrial animals. However, these drugs can lead to the development of resistant strains of bacteria. Shiitake mushrooms (SM) (Lentinula edodes) have long been popular as a health food in East Asia. Moreover, SM-derived polysaccharides are well-known as immunostimulants that possess antimicrobial properties. The aim of the present study was to evaluate the immunoprophylactic effects of SM against Bordetella bronchiseptica infection in mice as an initial step towards the development of eco-friendly feed additives to reduce the use of antimicrobials. Although SM had no effect on body weight gain under the un-infected conditions, SM alleviated progressive weight loss and helped in the recovery of body weight in B. bronchiseptica infected mice. Dietary supplementation with SM reinforced bacterial clearance in the infected mice. Of note, SM markedly increased the percentage of various T lymphocytes and the relative mRNA expression levels of tumor necrosis factor-α and interferon-γ in the bronchial lymph node early in the infection. Taken together, these findings suggest that SM could help in the improvement of body weight gain during B. bronchiseptica infection and may enhance the protective immune activity against a subclinical disease challenge, such as B. bronchiseptica infection in mice, probably by a strong stimulation of non-specific immune responses. Hence, SM may provide an alternative to reduce use of antimicrobials. Confirmation of the beneficial effects of SM as a feed additive is now required in industrial animals.

Immune-enhancing effect of fermented Maesil (Prunus mume Siebold & Zucc.) with probiotics against Bordetella bronchiseptica in mice.

Maesil (Prunus mume) has long been used as a traditional drug and healthy food in East Asian countries. It possesses a number of beneficial biological activities including potential antimicrobial effects against pathogens. Probiotics also have antibacterial effects. Moreover, some probiotics have an important role in regulating the immune system. The present study evaluated the immune enhancing effects of fermented Maesil with probiotics (Saccharomyces cerevisiae, Bacillus subtilis and Lactobacillus acidophilus) in mice, especially against Bordetella bronchiseptica, as an initial step towards the development of feed supplements for the promotion of immune activity and prevention of disease, especially in pigs. Continuous ingestion of fermented Maesil with probiotics markedly increased the macrophage ratio in peripheral blood and the T lymphocyte ratio in the spleen. In addition, antibody production against formalin-killed B. bronchiseptica significantly increased in the mice fed fermented Maesil compared with the control group. The number of leukocytes was significantly higher in the bronchio-alveolar lavage obtained from the fermented Maesil-fed animals compared to it in the control group at day 3 (maximal peak time) after experimental B. bronchiseptica infection. Moreover, at 7 day post-infection, relative messenger RNA expression levels of tumor necrosis factor- α and interferon-γ were significantly increased in splenocytes of mice fed fermented Maesil compared with those in the control group. Taken together, these findings suggest that feed containing fermented Maesil with probiotics enhances immune activity in mice, especially against B. bronchiseptica, via the potent stimulation of non-specific immune responses.

Measuring antimicrobial peptide activity on epithelial surfaces in cell culture.

To more accurately assess the activity and role of epithelial cell-derived antimicrobial peptides in their native settings, it is essential to perform assays at the surfaces under relevant conditions. In order to carry this out, we utilize three-dimensional cultures of airway and gingival epithelium, which are grown at an air-liquid interface. Under these conditions, the cultures can be subjected to challenge with a variety of factors known to cause an increase in antimicrobial peptide gene expression. The functional relevance of this induction can then be assessed by quantifying antibacterial activity either directly on the surface of the cells or using the fluid secreted onto the apical surface of the cultures. The relative contribution of the peptides can also be measured by pre-incubation of the secreted fluid with specific inhibitory antibodies. Thus, a relatively inexpensive in vitro model can be used to evaluate the role of antimicrobial peptides in mucosal epithelium.

Norepinephrine mediates acquisition of transferrin-iron in Bordetella bronchiseptica.

Previous research demonstrated that the sympathoadrenal catecholamine norepinephrine could promote the growth of Bordetella bronchiseptica in iron-restricted medium containing serum. In this study, norepinephrine was demonstrated to stimulate growth of this organism in the presence of partially iron-saturated transferrin but not lactoferrin. Although norepinephrine is known to induce transcription of the Bordetella bfeA enterobactin catechol xenosiderophore receptor gene, neither a bfeA mutant nor a bfeR regulator mutant was defective in growth responsiveness to norepinephrine. However, growth of a tonB mutant strain was not enhanced by norepinephrine, indicating that the response to this catecholamine was the result of high-affinity outer membrane transport. The B. bronchiseptica genome encodes a total of 19 known and predicted iron transport receptor genes, none of which, when mutated individually, were found to confer a defect in norepinephrine-mediated growth stimulation in the presence of transferrin. Labeling experiments demonstrated a TonB-dependent increase in cell-associated iron levels when bacteria grown in the presence of (55)Fe-transferrin were exposed to norepinephrine. In addition, TonB was required for maximum levels of cell-associated norepinephrine. Together, these results demonstrate that norepinephrine facilitates B. bronchiseptica iron acquisition from the iron carrier protein transferrin and this process may represent a mechanism by which some bacterial pathogens obtain this essential nutrient in the host environment.

Controlled release of Bordetella bronchiseptica dermonecrotoxin (BBD) vaccine from BBD-loaded chitosan microspheres in vitro.

Chitosan microspheres were prepared by ionic gelation process with sodium sulfate for nasal vaccine delivery. Bordetella Bronchiseptica Dermonecrotoxin (BBD) as a major virulence factor of a causative agent of atrophic rhinitis (AR) was loaded to the chitosan microspheres for vaccination. Morphology of BBD-loaded chitosan microspheres was observed as spherical shapes. The average particle sizes of the BBD-loaded chitosan microspheres were about 2.69 microm. More BBD was released with an increase of molecular weight of chitosan and with an increase of medium pH in vitro due to weaker intermolecular interaction between chitosan and BBD. Tumor necrosis factor-alpha (TNFalpha) and nitric oxide (NO) from RAW264.7 cells stimulated with BBD-loaded chitosan microspheres were gradually secreted, suggesting that released BBD from chitosan microspheres had immune stimulating activity of AR vaccine.

Role played by the response regulator Ris in Bordetella bronchiseptica resistance to macrophage killing.

Previous studies suggested that the persistence in eukaryotic cells of a Bordetella bronchiseptica mutant carrying an insertion in the locus encoding the response regulator RisAS is impaired. This suggested that ris-dependent products are required for the intracellular survival of bacteria. In this study we demonstrate that ris-regulated products play a role in B. bronchiseptica resistance against both phagosomal acidification and reactive oxygen intermediates.

Disruption of tonB in Bordetella bronchiseptica and Bordetella pertussis prevents utilization of ferric siderophores, haemin and haemoglobin as iron sources.

The Bordetella bronchiseptica tonB gene was cloned by detection of a chromosomal restriction fragment hybridizing with each of two degenerate oligonucleotides that corresponded to Pro-Glu and Pro-Lys repeats characteristic of known TonB proteins. The tonB(Bb) gene was situated upstream of exbB and exbD homologues and downstream of a putative Fur-regulated promoter. Hybridization results indicated that the tonB operon and flanking regions were highly conserved between B. bronchiseptica, Bordetella pertussis and Bordetella parapertussis. Disruption of tonB in B. bronchiseptica resulted in inability to grow in iron-limiting media, and inability to utilize alcaligin, enterobactin, ferrichrome, desferroxamine B, haemin and haemoglobin. Although it was not possible to inactivate tonB in a clinical B. pertussis isolate, tonB was disrupted in a laboratory B. pertussis strain previously selected for the ability to grow on Luria-Bertani medium. This B. pertussis tonB mutant shared a similar iron complex utilization deficient phenotype with the B. bronchiseptica tonB mutant. The B. bronchiseptica tonB operon present on a plasmid did not complement an Escherichia coli tonB mutant, but inefficient reconstitution of enterobactin utilization was observed in one fepA mutant harbouring plasmid copies of the B. pertussis fepA homologue and tonB(Bb) operon.

The lipopolysaccharide of Bordetella bronchiseptica acts as a protective shield against antimicrobial peptides.

Resistance profiles of the two Bordetella species B. bronchiseptica and B. pertussis against various antimicrobial peptides were determined in liquid survival and agar diffusion assays. B. bronchiseptica exhibited significantly higher resistance against all tested peptides than B. pertussis. The most powerful agents acting on B. bronchiseptica were, in the order of their killing efficiencies, cecropin P > cecropin B > magainin-II-amide > protamine > melittin. Interestingly, for B. bronchiseptica, the resistance level was significantly affected by phase variation, as a bvgS deletion derivative showed an increased sensitivity to these peptides. Tn5-induced protamine-sensitive B. bronchiseptica mutants, which were found to be very susceptible to most of the cationic peptides, were isolated. In two of these mutants, the genetic loci inactivated by transposon insertion were identified as containing genes highly homologous to the wlbA and wlbL genes of B. pertussis that are involved in the biosynthesis of lipopolysaccharide (LPS). In agreement with this finding, the two peptide-sensitive mutants revealed structural changes in the LPS, resulting in the loss of the O-specific side chains and the prevalence of the LPS core structure. This demonstrates that LPS plays a major role in the resistance of B. bronchiseptica against the action of antimicrobial peptides and suggests that B. pertussis is much more susceptible to these peptides due to the lack of the highly charged O-specific sugar side chains.

Phase variation affects long-term survival of Bordetella bronchiseptica in professional phagocytes.

Several Bordetella bronchiseptica isolates were investigated for intracellular survival in macrophages. A significant number of viable bacteria of all strains could be recovered even after 96 h postinfection. In all cases bvg mutants of the B. bronchiseptica strains showed a significant survival advantage over the respective wild-type strains. The bacteria were already located in phagolysosomes early after uptake. Neither opsonization of the bacteria nor activation of the macrophages with gamma interferon or lipopolysaccharide prior to infection affected uptake and survival of the bacteria.

An iron-regulated outer-membrane protein specific to Bordetella bronchiseptica and homologous to ferric siderophore receptors.

The bfrA (Bordetella bronchiseptica ferric iron repressed outer-membrane protein) gene was cloned from Bordetella bronchiseptica by screening a library of TnphoA insertion mutants for iron-repressed fusions to phoA. The bfrA gene encoded an 80 kDa outer-membrane protein with a high level of amino acid sequence identity to several bacterial proteins belonging to the family of Ton B-dependent outer-membrane receptors. BfrA was especially homologous to Cir of Escherichia coli, IrgA of Vibrio cholerae and to three previously characterized ferric enterobactin receptors. DNA hybridization results indicated that bfrA was not present in other Bordetella species. Expression of the bfrA gene was induced by low iron availability from a promoter overlapped by a sequence resembling a consensus Fur-binding sequence, and bfrA expression was derepressed in a B. bronchiseptica fur mutant. Utilization of the Bordetella siderophore alcaligin and the exogenous siderophore enterobactin was unaffected in bfrA mutants. Upon attempting to find the specificity of BfrA, 2,3-dihydroxybenzoylserine (DHBS) was shown to be utilized in a bfeA (Bordetella ferric enterobactin receptor gene)-dependent manner by B. bronchiseptica and B. pertussis. In addition, the hydroxamate siderophores ferrichrome and desferrioxamine B, and the iron source haemin were shown to be utilized independently of bfeA and bfrA in B. bronchiseptica and B. pertussis.

Influence of culture conditions on expression of antigens in Bordetella bronchiseptica.

Bordetella (B.) bronchiseptica isolates from the respiratory tract of rat and pig in their virulent phase-I and their spontaneously developed avirulent phase-III were investigated. The strains were cultured on Bordet-Gengou agar, on nutrition agar with 10% horse blood, and with 5% sheep blood, on yeast agar, on minimal nutrition agar (MM) and Simmon's citrate agar with glycerol, starch, and nicotinic acid. Antigenic modulation was induced by MgSO4 on Bordet-Gengou agar. The B. bronchiseptica strains were cultivated at 37 degrees C for 48 h. The influence of different MgSO4 concentrations after five passages (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100 mM/ml and low (20 degrees C) and high (42 degrees C) temperatures on antigenic modulation was investigated on Bordet-Gengou (B-G) agar. B. bronchiseptica colonies were characterized in relation to morphology, haemolysis, slide agglutination with B. bronchiseptica phase-I polyclonal antibodies, haemagglutination with horse and calf erythrocytes and polypeptide and LPS patterns in SDS-PAGE. Cultivation of B. bronchiseptica phase-I on B-G agar and ACMM agar at 37 degrees C for 48 h resulted in strong phase-I antigenic patterns, and, on peptone-rich media, in antigenic modulation. The morphology of B. bronchiseptica phase-I-strain colonies on peptone media was different from that of B-G and ACMM agar. The LPS pattern of both strains resembled that of phase-III strains. MgSO4 concentrations of 1 mM/ml (strain 1636 I) and 3 mM/ml (Ratte I) were able to induce LPS-pattern-like phase III.