Age-Dependent Effects of Type I and Type III IFNs in the Pathogenesis of Bordetella pertussis Infection and Disease.

Type I and III IFNs play diverse roles in bacterial infections, being protective for some but deleterious for others. Using RNA-sequencing transcriptomics we investigated lung gene expression responses to Bordetella pertussis infection in adult mice, revealing that type I and III IFN pathways may play an important role in promoting inflammatory responses. In B. pertussis-infected mice, lung type I/III IFN responses correlated with increased proinflammatory cytokine expression and with lung inflammatory pathology. In mutant mice with increased type I IFN receptor (IFNAR) signaling, B. pertussis infection exacerbated lung inflammatory pathology, whereas knockout mice with defects in type I IFN signaling had lower levels of lung inflammation than wild-type mice. Curiously, B. pertussis-infected IFNAR1 knockout mice had wild-type levels of lung inflammatory pathology. However, in response to infection these mice had increased levels of type III IFN expression, neutralization of which reduced lung inflammation. In support of this finding, B. pertussis-infected mice with a knockout mutation in the type III IFN receptor (IFNLR1) and double IFNAR1/IFNLR1 knockout mutant mice had reduced lung inflammatory pathology compared with that in wild-type mice, indicating that type III IFN exacerbates lung inflammation. In marked contrast, infant mice did not upregulate type I or III IFNs in response to B. pertussis infection and were protected from lethal infection by increased type I IFN signaling. These results indicate age-dependent effects of type I/III IFN signaling during B. pertussis infection and suggest that these pathways represent targets for therapeutic intervention in pertussis.

Specific bacteriophage of Bordetella bronchiseptica regulates B. bronchiseptica-induced microRNA expression profiles to decrease inflammation in swine nasal turbinate cells.

BACKGROUND: Respiratory diseases in pigs are the main health concerns for swine producers. Similar to the diseases in human and other animals, respiratory diseases are primary related to morbidity and are the result of infection with bacteria, viruses, or both. B. bronchiseptica causes serious respiratory diseases in the swine airway track. However, the B. bronchiseptica-specific bacteriophage has diverse advantages such as decreasing antibiotic overuse and possible therapeutic potential against bacteria. OBJECTIVE: The objects of this study were to investigate the therapeutic effect of specific B. bronchiseptica bacteriophages and to identify genes related to bacteriophage signaling utilizing RNA microarrays in swine nasal turbinate cells. METHODS: Bor-BRP-1 phages were applied 24 h prior to B.bronchiseptica infection (1 × 107 cfu/ml) at several concentrations of bacterial infection. Cells were incubated to detect cytokines and 24 h to detect mucin production. And real-time quantitative PCR was performed to examine related genes expression. To determine the change of total gene expression based on B.bronchiseptica and Bor-BRP-1 treatment, we performed RNA sequencing experiments. RESULTS: The results showed that B. bronchiseptica induced increased expression of several inflammatory genes such as IL-1ß, IL-6, and Muc1 in a dose-dependent manner. However, Bor-BRP-1 induced reduction of gene expression compared to the B. bronchiseptica induction group. In addition, microarrays detected Bor-BRP-1-altered inflammatory gene expression against B. bronchiseptica, reducing B. bronchiseptica-induced airway inflammation in swine epithelial cells. CONCLUSION: These results suggest that the specific bacteriophage has a therapeutic potential to defend against B. bronchiseptica infection by altering inflammatory gene expression profiles.

Bordetella bronchiseptica Diguanylate Cyclase BdcA Regulates Motility and Is Important for the Establishment of Respiratory Infection in Mice.

Bacteria can be motile and planktonic or, alternatively, sessile and participating in the biofilm mode of growth. The transition between these lifestyles can be regulated by a second messenger, cyclic dimeric GMP (c-di-GMP). High intracellular c-di-GMP concentration correlates with biofilm formation and motility inhibition in most bacteria, including Bordetella bronchiseptica, which causes respiratory tract infections in mammals and forms biofilms in infected mice. We previously described the diguanylate cyclase BdcA as involved in c-di-GMP synthesis and motility regulation in B. bronchiseptica; here, we further describe the mechanism whereby BdcA is able to regulate motility and biofilm formation. Amino acid replacement of GGDEF with GGAAF in BdcA is consistent with the conclusion that diguanylate cyclase activity is necessary for biofilm formation and motility regulation, although we were unable to confirm the stability of the mutant protein. In the absence of the bdcA gene, B. bronchiseptica showed enhanced motility, strengthening the hypothesis that BdcA regulates motility in B. bronchiseptica We showed that c-di-GMP-mediated motility inhibition involved regulation of flagellin expression, as high c-di-GMP levels achieved by expressing BdcA significantly reduced the level of flagellin protein. We also demonstrated that protein BB2109 is necessary for BdcA activity, motility inhibition, and biofilm formation. Finally, absence of the bdcA gene affected bacterial infection, implicating BdcA-regulated functions as important for bacterium-host interactions. This work supports the role of c-di-GMP in biofilm formation and motility regulation in B. bronchiseptica, as well as its impact on pathogenesis.IMPORTANCE Pathogenesis of Bordetella spp., like that of a number of other pathogens, involves biofilm formation. Biofilms increase tolerance to biotic and abiotic factors and are proposed as reservoirs of microbes for transmission to other organs (trachea, lungs) or other hosts. Bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is a second messenger that regulates transition between biofilm and planktonic lifestyles. In Bordetella bronchiseptica, high c-di-GMP levels inhibit motility and favor biofilm formation. In the present work, we characterized a B. bronchiseptica diguanylate cyclase, BdcA, which regulates motility and biofilm formation and affects the ability of B. bronchiseptica to colonize the murine respiratory tract. These results provide us with a better understanding of how B. bronchiseptica can infect a host.

Bacterial and Pneumocystis Infections in the Lungs of Gene-Knockout Rabbits with Severe Combined Immunodeficiency.

Using the CRISPR/Cas9 gene-editing technology, we recently produced a number of rabbits with mutations in immune function genes, including FOXN1, PRKDC, RAG1, RAG2, and IL2RG. Seven founder knockout rabbits (F0) and three male IL2RG null (-/y) F1 animals demonstrated severe combined immunodeficiency (SCID), characterized by absence or pronounced hypoplasia of the thymus and splenic white pulp, and absence of immature and mature T and B-lymphocytes in peripheral blood. Complete blood count analysis showed severe leukopenia and lymphocytopenia accompanied by severe neutrophilia. Without prophylactic antibiotics, the SCID rabbits universally succumbed to lung infections following weaning. Pathology examination revealed severe heterophilic bronchopneumonia caused by Bordetella bronchiseptica in several animals, but a consistent feature of lung lesions in all animals was a severe interstitial pneumonia caused by Pneumocystis oryctolagi, as confirmed by histological examination and PCR analysis of Pneumocystis genes. The results of this study suggest that these SCID rabbits could serve as a useful model for human SCID to investigate the disease pathogenesis and the development of gene and drug therapies.

Host Specificity of Ovine Bordetella parapertussis and the Role of Complement.

The classical bordetellae are comprised of three subspecies that differ from broad to very limited host specificity. Although several lineages appear to have specialized to particular host species, most retain the ability to colonize and grow in mice, providing a powerful common experimental model to study their differences. One of the subspecies, Bordetella parapertussis, is composed of two distinct clades that have specialized to different hosts: one to humans (Bpphu), and the other to sheep (Bppov). While Bpphu and the other classical bordetellae can efficiently colonize mice, Bppov strains are severely defective in their ability to colonize the murine respiratory tract. Bppov genomic analysis did not reveal the loss of adherence genes, but substantial mutations and deletions of multiple genes involved in the production of O-antigen, which is required to prevent complement deposition on B. bronchiseptica and Bpphu strains. Bppov lacks O-antigen and, like O-antigen mutants of other bordetellae, is highly sensitive to murine complement-mediated killing in vitro. Based on these results, we hypothesized that Bppov failed to colonize mice because of its sensitivity to murine complement. Consistent with this, the Bppov defect in the colonization of wild type mice was not observed in mice lacking the central complement component C3. Furthermore, Bppov strains were highly susceptible to killing by murine complement, but not by sheep complement. These data demonstrate that the failure of Bppov to colonize mice is due to sensitivity to murine, but not sheep, complement, providing a mechanistic example of how specialization that accompanies expansion in one host can limit host range.

Flagellin typing of Bordetella bronchiseptica strains originating from different host species.

Bordetella bronchiseptica is a widespread Gram-negative pathogen occurring in different mammal species. It is known to play a role in the aetiology of infectious atrophic rhinitis of swine, canine kennel cough, respiratory syndromes of cats, rabbits and guinea pigs, and sporadic human cases have also been reported. In this study, 93 B. bronchiseptica strains were examined from a broad range of host species and different geographical regions using restriction fragment length polymorphism analysis of polymerase chain reaction products of flaA to reveal the possible host-specificity of the flagellin. Eight types (A-H) of flaA were identified, including five newly described ones (D-H). All but one of the 22 B. bronchiseptica strains from swine showed type B fragment pattern. The eighteen Hungarian isolates of canine origin were uniform (type A) while in other countries type B and D were also present in dogs. The sequence and phylogenetic analysis of 36 representative strains of flaA types revealed four clusters. These clusters correlated with flaA PCR-RFLP types and host species, especially in pigs and dogs. The revealed diversity of the strains isolated from human cases indicated possible zoonotic transmissions from various animal sources.

Molecular signatures of the evolving immune response in mice following a Bordetella pertussis infection.

Worldwide resurgence of pertussis necessitates the need for improvement of pertussis vaccines and vaccination strategies. Since natural infections induce a longer-lasting immunity than vaccinations, detailed knowledge of the immune responses following natural infection can provide important clues for such improvement. The purpose was to elucidate the kinetics of the protective immune response evolving after experimental Bordetella pertussis (B. pertussis) infection in mice. Data were collected from (i) individual analyses, i.e. microarray, flow cytometry, multiplex immunoassays, and bacterial clearance; (ii) twelve time points during the infection; and (iii) different tissues involved in the immune responses, i.e. lungs, spleen and blood. Combined data revealed detailed insight in molecular and cellular sequence of events connecting different phases (innate, bridging and adaptive) of the immune response following the infection. We detected a prolonged acute phase response, broad pathogen recognition, and early gene signatures of subsequent T-cell recruitment in the lungs. Activation of particular transcription factors and specific cell markers provided insight into the time course of the transition from innate towards adaptive immune responses, which resulted in a broad spectrum of systemic antibody subclasses and splenic Th1/Th17 memory cells against B. pertussis. In addition, signatures preceding the local generation of Th1 and Th17 cells as well as IgA in the lungs, considered key elements in protection against B. pertussis, were established. In conclusion, molecular and cellular immunological processes in response to live B. pertussis infection were unraveled, which may provide guidance in selecting new vaccine candidates that should evoke local and prolonged protective immune responses.

Comparative analyses of a cystic fibrosis isolate of Bordetella bronchiseptica reveal differences in important pathogenic phenotypes.

Bordetella bronchiseptica is a Gram-negative bacterium that infects and causes disease in a wide variety of animals. B. bronchiseptica also infects humans, thereby demonstrating zoonotic transmission. An extensive characterization of human B. bronchiseptica isolates is needed to better understand the distinct genetic and phenotypic traits associated with these zoonotic transmission events. Using whole-genome transcriptome and CGH analysis, we report that a B. bronchiseptica cystic fibrosis isolate, T44625, contains a distinct genomic content of virulence-associated genes and differentially expresses these genes compared to the sequenced model laboratory strain RB50, a rabbit isolate. The differential gene expression pattern correlated with unique phenotypes exhibited by T44625, which included lower motility, increased aggregation, hyperbiofilm formation, and an increased in vitro capacity to adhere to respiratory epithelial cells. Using a mouse intranasal infection model, we found that although defective in establishing high bacterial burdens early during the infection process, T44625 persisted efficiently in the mouse nose. By documenting the unique genomic and phenotypic attributes of T44625, this report provides a blueprint for understanding the successful zoonotic potential of B. bronchiseptica and other zoonotic bacteria.

[The kit of reagents for polymerase chain reaction diagnostic of infections caused by B. pertussis, B. parapertussis and B. bronchiseptica].

The effective treatment of whooping cough directly depends of early diagnostics. The polymerase chain reaction diagnostic is the most perspective diagnostic technique. The kit of reagents is developed to diagnose whooping cough, parapertussis and bronchosepticosis with polymerase chain reaction. The evaluation of its analytical characteristics was carried out. The sensitivity made 1 x 103 of genome equivalents per 1 ml of sample (the sorption technique of DNA extraction was applied) and 5 x 102 of genome equivalents per 1 ml (the precipitation technique of DNA extraction was used). The specificity of test in the framework of analyzed panel of strains and isolates of microorganisms made 100%. The diagnostic sensitivity of analysis exceeded the sensitivity of bacteriological analysis up to 20 times. The application of this kit of reagents permits to detect and to differentiate DNA of agent of whooping cough, parapertussis during one working day already at the beginning of catarrhal period of disease and up to 18th day from the moment of cough appearance. In perspective, this process creates an opportunity to apply timely the specific therapy. The specter of agents of acute respiratory diseases brining on acute prolonged cough in children who were directed to bacteriological analysis to confirm whooping cough is investigated.

Swine atrophic rhinitis caused by pasteurella multocida toxin and bordetella dermonecrotic toxin.

Atrophic rhinitis is a widespread and economically important swine disease caused by Pasteurella multocida and Bordetella bronchiseptica. The disease is characterized by atrophy of the nasal turbinate bones, which results in a shortened and deformed snout in severe cases. P. multocida toxin and B. bronchiseptica dermonecrotic toxin have been considered to independently or cooperatively disturb the osteogenesis of the turbinate bone by inhibiting osteoblastic differentiation and/or stimulating bone resorption by osteoclasts. Recently, the intracellular targets and molecular actions of both toxins have been clarified, enabling speculation on the intracellular signals leading to the inhibition of osteogenesis.

Immune regulation of a chronic bacteria infection and consequences for pathogen transmission.

BACKGROUND: The role of host immunity has been recognized as not only playing a fundamental role in the interaction between the host and pathogen but also in influencing host infectiousness and the ability to shed pathogens. Despite the interest in this area of study, and the development of theoretical work on the immuno-epidemiology of infections, little is known about the immunological processes that influence pathogen shedding patterns. RESULTS: We used the respiratory bacterium Bordetella bronchiseptica and its common natural host, the rabbit, to examine the intensity and duration of oro-nasal bacteria shedding in relation to changes in the level of serum antibodies, blood cells, cytokine expression and number of bacteria colonies in the respiratory tract. Findings show that infected rabbits shed B. bronchiseptica by contact up to 4.5 months post infection. Shedding was positively affected by number of bacteria in the nasal cavity (CFU/g) but negatively influenced by serum IgG, which also contributed to the initial reduction of bacteria in the nasal cavity. Three main patterns of shedding were identified: i- bacteria were shed intermittently (46% of individuals), ii- bacteria shedding fell with the progression of the infection (31%) and iii- individuals never shed bacteria despite being infected (23%). Differences in the initial number of bacteria shed between the first two groups were associated with differences in the level of serum antibodies and white blood cells. These results suggest that the immunological conditions at the early stage of the infection may play a role in modulating the long term dynamics of B. bronchiseptica shedding. CONCLUSIONS: We propose that IgG influences the threshold of bacteria in the oro-nasal cavity which then affects the intensity and duration of individual shedding. In addition, we suggest that a threshold level of infection is required for shedding, below this value individuals never shed bacteria despite being infected. The mechanisms regulating these interactions are still obscure and more studies are needed to understand the persistence of bacteria in the upper respiratory tract and the processes controlling the intensity and duration of shedding.

Comparative role of immunoglobulin A in protective immunity against the Bordetellae.

The genus Bordetella includes a group of closely related mammalian pathogens that cause a variety of respiratory diseases in a long list of animals (B. bronchiseptica) and whooping cough in humans (B. pertussis and B. parapertussis). While past research has examined how these pathogens are eliminated from the lower respiratory tract, the host factors that control and/or clear the bordetellae from the upper respiratory tract remain unclear. We hypothesized that immunoglobulin A (IgA), the predominant mucosal antibody isotype, would have a protective role against these mucosal pathogens. IgA(-/-) mice were indistinguishable from wild-type mice in their control and clearance of B. pertussis or B. parapertussis, suggesting that IgA is not crucial to immunity to these organisms. However, naïve and convalescent IgA(-/-) mice were defective in reducing the numbers of B. bronchiseptica in the upper respiratory tract compared to wild-type controls. Passively transferred serum from convalescent IgA(-/-) mice was not as effective as serum from convalescent wild-type mice in clearing this pathogen from the tracheae of naive recipient mice. IgA induced by B. bronchiseptica infection predominantly recognized lipopolysaccharide-containing O-antigen, and antibodies against O-antigen were important to bacterial clearance from the trachea. Since an IgA response contributes to the control of B. bronchiseptica infection of the upper respiratory tract, immunization strategies aimed at inducing B. bronchiseptica-specific IgA may be beneficial to preventing the spread of this bacterium among domestic animal populations.

Role of Bordetella bronchiseptica adenylate cyclase in nasal colonization and in development of local and systemic immune responses in piglets.

Two Bordetella bronchiseptica mutants, lacking the adenylate cyclase (Cya) or both Cya and pertactin (Prn), were compared with their parental strain NL1013 in their abilities to colonize the nose of neonate piglets and to induce local and systemic antibody responses against filamentous hemagglutinin (FHA) after intranasal (i.n.) inoculation. The number of bacteria recovered and the duration of infection in the nasal secretions were greater for the wild-type parent strain than for the Cya-deficient mutant, indicating that Cya plays an important role during B. bronchiseptica colonization of the nasal cavity. The double mutant did not colonize the nasal cavity and was less able to adhere to epithelial cells in vitro than the other two strains, supporting the hypothesis that Prn plays a major role in cell adhesion. In piglets inoculated with the wild type strain, anti-FHA IgM was found in the nasal secretions one week after inoculation, followed two weeks later by anti-FHA IgA; their presence was concomitant with decreases in bacterial counts. Anti-FHA IgG appeared at six weeks after infection in the serum. In contrast, i.n. inoculation with either mutant failed to induce a nasal secretory antibody response but did induce an earlier and higher IgM response in the serum than inoculation with the wild type strain. However, only the Cya-deficient mutant was able to prime the piglets for the development of a secondary nasal IgM and serum IgG response to FHA after intranasal inoculation with the wild type B. bronchiseptica.

Toll-like receptor 4-mediated innate IL-10 activates antigen-specific regulatory T cells and confers resistance to Bordetella pertussis by inhibiting inflammatory pathology.

Signaling through Toll-like receptors (TLR) activates dendritic cell (DC) maturation and IL-12 production, which directs the induction of Th1 cells. We found that the production of IL-10, in addition to inflammatory cytokines and chemokines, was significantly reduced in DCs from TLR4-defective C3H/HeJ mice in response to Bordetella pertussis. TLR4 was also required for B. pertussis LPS-induced maturation of DCs, but other B. pertussis components stimulated DC maturation independently of TLR4. The course of B. pertussis infection was more severe in C3H/HeJ than in C3H/HeN mice. Surprisingly, Ab- and Ag-specific IFN-gamma responses were enhanced at the peak of infection, whereas Ag-specific IL-10-producing T cells were significantly reduced in C3H/HeJ mice. This was associated with enhanced inflammatory cytokine production, cellular infiltration, and severe pathological changes in the lungs of TLR4-defective mice. Our findings suggest that TLR-4 signaling activates innate IL-10 production in response to B. pertussis, which both directly, and by promoting the induction of IL-10-secreting type 1 regulatory T cells, may inhibit Th1 responses and limit inflammatory pathology in the lungs during infection with B. pertussis.

Genetic variation in response of turkeys to experimental infection with Bordetella avium.

One hundred ninety-six male and female turkeys representing two genetic lines were experimentally infected with Bordetella avium. The lines of turkeys included a randombred control line (RBC2) and a subline (F) of RBC2 selected for increased 16-wk body weight. No difference was found between lines RBC2 and F in the number of days to onset of clinical signs, and no mortality due to B. avium infection was observed in either line. Interestingly, however, a significant depression (12%) occurred in body weight of F line poults infected with B. avium, but no significant depression occurred in body weight of RBC2 poults.

In vivo and in vitro analysis of Bordetella pertussis catalase and Fe-superoxide dismutase mutants.

Bordetella pertussis produces a catalase and a Fe-superoxide dismutase. The importance of these enzymes in virulence was investigated, in vitro as well as in vivo, by using mutants deficient in their production. The catalase-deficient mutant survived within polymorphonuclear leukocytes, killed J774A.1 macrophages through apoptosis, and behaved as the parental strain in a murine respiratory infection model. These results suggest no direct role for catalase in B. pertussis virulence. The absence of expression of Fe-superoxide dismutase had profound effects on the bacterium including a reduced ability to express adenylate cyclase-hemolysin and pertactin, two factors important for B. pertussis pathogenesis. The Fe-superoxide dismutase-deficient mutant also had decreased abilities to colonize and persist in the murine respiratory infection model.

Characterization of IS1001, an insertion sequence element of Bordetella parapertussis.

By analysis of repetitive DNA in Bordetella parapertussis, an insertion sequence element, designated IS1001, was identified. Sequence analysis revealed that IS1001 comprised 1,306 bp and contained inverted repeats at its termini. Furthermore, several open reading frames that may code for transposition functions were identified. The largest open reading frame coded for a protein comprising 406 amino acid residues and showed homology to TnpA, which is encoded by an insertion sequence element (IS1096) found in Mycobacterium smegmatis. Examination of flanking sequences revealed that insertion of IS1001 occurs preferentially in stretches of T's or A's and results in a duplication of target sequences of 6 to 8 bases. IS1001 was found in about 20 copies in 10 B. parapertussis strains analyzed. No restriction fragment length polymorphism was observed in B. parapertussis when IS1001 was used as a probe. An insertion sequence element similar or identical to IS1001 was found in B. bronchiseptica strains isolated from pigs and a rabbit. In these strains, about five copies of the IS1001-like element were present at different positions in the bacterial chromosome. Neither B. pertussis nor B. bronchiseptica strains isolated from humans and dogs contained an IS1001-like element. Therefore, IS1001 may be used as a specific probe for the detection of B. parapertussis in human clinical samples.