Adherence of Bordetella bronchiseptica 276 to porcine trachea maintained in organ culture.

Two organ culture models have been adapted for porcine tracheae in order to study colonization by Bordetella bronchiseptica. Rings or segments excised from tracheae of newborn piglets were incubated overnight at 37 degrees C in a nutrient medium under 5% CO2-95% air conditions. Tracheal segments were infected with B bronchiseptica 276, and after different incubation times, bacterial counts were done. B. bronchiseptica adhered well to tracheae maintained in culture, and no statistically significant differences between the two models were observed. Noninfected tracheal mucosae maintained a normal appearance for several days, whereas infected mucosae showed typical damage caused by B. bronchiseptica, namely, loss of ciliary activity and cilia and sloughing of ciliated cells. Our data indicated that porcine tracheal organ culture could be advantageously used to study in vitro colonization by B. bronchiseptica.     

Electron microscopic observations on ciliated epithelium of tracheal organ cultures infected with Bordetella bronchiseptica

Using mouse tracheal organ cultures, the pathogenic effect of Bordetella bronchiseptica to epithelial cells was studied by electron microscopy. The ultrastructure of epithelial cells in uninfected tracheal rings was preserved well for longer than 3 days. In mouse tracheal rings infected with graded doses (3 x 10(5) to 10(7) CFU/ml) of phase I B. bronchiseptica, the colonization in the interciliary spaces of ciliated epithelial cells was observed after a 20-hr infection period. The infected tracheal rings showed swelling of nonciliated cells as well as ciliated cells, rupture of cell membrane of cilia, swelling and disappearance of cilia, and atrophic cytomorphosis of epithelial cells. The severity of these changes occurred depending on the infection doses. These changes were essentially similar to those observed previously in the tracheal epithelia of the B. bronchiseptica-infected mice. The usefulness of this in vitro model was suggested for studying the pathogenesis of Bordetella infection.     

Bordetella type III secretion modulates dendritic cell migration resulting in immunosuppression and bacterial persistence

Chronic bacterial infection reflects a balance between the host immune response and bacterial factors that promote colonization and immune evasion. Bordetella bronchiseptica uses a type III secretion system (TTSS) to persist in the lower respiratory tract of mice. We hypothesize that colonization is facilitated by bacteria-driven modulation of dendritic cells (DCs), which leads to an immunosuppressive adaptive host response. Migration of DCs to the draining lymph nodes of the respiratory tract was significantly increased in mice infected with wild-type B. bronchiseptica compared with mice infected with TTSS mutant bacteria. Reduced colonization by TTSS-deficient bacteria was evident by 7 days after infection, whereas colonization by wild-type bacteria remained high. This decrease in colonization correlated with peak IFN-gamma production by restimulated splenocytes from infected animals. Wild-type bacteria also elicited peak IFN-gamma production on day 7, but the quantity was significantly lower than that elicited by TTSS mutant bacteria. Additionally, wild-type bacteria elicited higher levels of the immunosuppressive cytokine IL-10 compared with the TTSS mutant bacteria. B. bronchiseptica colonization in IL-10(-/-) mice was significantly reduced compared with infections in wild-type mice. These findings suggest that B. bronchiseptica use the TTSS to rapidly drive respiratory DCs to secondary lymphoid tissues where these APCs stimulate an immunosuppressive response characterized by increased IL-10 and decreased IFN-gamma production that favors bacterial persistence.     

Electron microscopic observations on tracheal epithelia of mice infected with Bordetella bronchiseptica

To clarify the pathogenesis of Bordetella in vivo infection, the tracheal epithelia of mice were examined in detail by electron microscopy at various intervals after intranasal inoculation with graded doses of phase I Bordetella bronchiseptica. In mice infected with a lethal dose (6 to 7 x 10(7) CFU), a remarkable rupture of the cell membranes of cilia and microvilli of the middle trachea was found on day I postinfection. The rupture of the membrane was observed over the entire tracheal epithelia, on day 2 after infection. The affected cilia were constricted at the transitional region and were broken off. In the ciliated cells the adherence of organisms to ciliary apexes and colonization in the interciliary spaces were also remarkable. In both the ciliated and nonciliated epithelial cells, the cytoplasmic vacuolation and pyknosis or karyorrehexis were also notable. In mice infected with one-tenth of the lethal dose, similar findings were seen, but appeared more slowly and the bacteria were not seen attaching to ciliary apexes. In mice receiving one-hundredth of the lethal dose, only mild cilial abnormality such as aggregation of cilia, and slight cytoplasmic vacuolation were found 6 days postinfection. Based on these findings, a possible mechanism of the ciliary damages produced by B. bronchiseptica was postulated.     

Bordetella type III secretion and adenylate cyclase toxin synergize to drive dendritic cells into a semimature state

Bordetella bronchiseptica establishes persistent infection of the murine respiratory tract. We hypothesize that long-term colonization is mediated in part by bacteria-driven modulation of dendritic cells (DCs) leading to altered adaptive immune responses. Bone marrow-derived DCs (BMDCs) from C57BL/6 mice infected with live B. bronchiseptica exhibited high surface expression of MHCII, CD86, and CD80. However, B. bronchiseptica-infected BMDCs did not exhibit significant increases in CD40 surface expression and IL-12 secretion compared with BMDCs treated with heat-killed B. bronchiseptica. The B. bronchiseptica type III secretion system (TTSS) mediated the increase in MHCII, CD86, and CD80 surface expression, while the inhibition of CD40 and IL-12 expression was mediated by adenylate cyclase toxin (ACT). IL-6 secretion was independent of the TTSS and ACT. These phenotypic changes may result from differential regulation of MAPK signaling in DCs. Wild-type B. bronchiseptica activated the ERK 1/2 signaling pathway in a TTSS-dependent manner. Additionally, ACT was found to inhibit p38 signaling. These data suggest that B. bronchiseptica drive DC into a semimature phenotype by altering MAPK signaling. These semimature DCs may induce tolerogenic immune responses that allow the persistent colonization of B. bronchiseptica in the host respiratory tract.     

Bordetella bronchiseptica PagP is a Bvg-regulated lipid A palmitoyl transferase that is required for persistent colonization of the mouse respiratory tract

Bordetella bronchiseptica lipopolysaccharide (LPS) expression varies depending on growth conditions, regulated by the Bvg system. A B. bronchiseptica pagP homologue was identified that is required for Bvg-mediated modification of the lipid A core region of LPS that occurs on switching from the Bvg- to the Bvg+ phase. Structural analysis demonstrated that the lipid A of a B. bronchiseptica pagP mutant differed from wild-type lipid A by the absence of a palmitate group in secondary acylation at the C3' position. The putative pagP promoter drove the expression of a green fluorescent protein (GFP) reporter gene in a Bvg-regulated fashion. These data suggest that B. bronchiseptica pagP encodes a Bvg-regulated lipid A palmitoyl transferase that mediates modification of the lipid A as part of the overall Bvg-mediated adaptation of this organism to changing environmental conditions. We also show that pagP is not required for the initial colonization of the mouse respiratory tract by B. bronchiseptica, but is required for persistence of the organism within this organ.     

Suppression of NF-kappaB-mediated beta-defensin gene expression in the mammalian airway by the Bordetella type III secretion system

Expression of innate immune genes such as beta-defensins is induced in airway epithelium by bacterial components via activation of NF-kappaB. We show here that live Gram-negative bacteria can similarly stimulate this pathway, resulting in upregulation of the beta-defensin tracheal antimicrobial peptide (TAP) in primary cultures of bovine tracheal epithelial cells (TECs), by a Toll-like receptor 4 (TLR4)-mediated pathway. The Gram-negative airway pathogen Bordetella bronchiseptica possesses a type III secretion system previously suggested to inhibit the nuclear translocation of NF-kappaB in a cell line by immunohistochemistry. We therefore hypothesized that this pathogen might interfere in the innate immune response of the epithelium. Exposure of TECs to wild-type B. bronchiseptica suppressed the activation of NF-kappaB and the subsequent induction of TAP mRNA levels, whereas a type III secretion-defective strain did not. These results suggest a mechanism for bacterial evasion of the innate immune response in the airway, which could allow for the observed persistent colonization of this pathogen.     

Scanning electron microscopy of mouse ciliated oviduct and tracheal epithelium infected in vitro with Bordetella pertussis

Infection of mouse tracheal organ culture with Bordetella pertussis resulted in ciliostasis within 36 h. Scanning electron microscopy revealed that B. pertussis attached exclusively to ciliated cells but did not induce expulsion of this cell type at a test interval of 48 h. Mouse oviduct organ culture infected with B. pertussis demonstrated the same strict tropism for ciliated cells as in the tracheal ring system. Only ciliated cells were parasitized, becoming heavily colonized 48 h postinfection. Infected ciliated oviduct cells were not extruded. A fixation method which enhances fine structure was used in the scanning electron microscope studies. Bacterial fimbriae were not observed as the method of attachment of B. pertussis to cilia but fine fibers were seen extending between cilia and bacterial cells.     

Efficacy of commercial vaccines for protecting guinea pigs against Bordetella bronchiseptica pneumonia

Autogenous bacterins are recommended to protect guinea pigs (Cavia porcellus) against pneumonia due to Bordetella bronchiseptica. Bordetella vaccines are available commercially for several other animal species. The substantial antigenic cross-reactivity among Bordetella isolates from various animal species suggests that immunity resulting from use of these vaccines might protect guinea pigs. Groups of ten individually housed Hartley guinea pigs from a colony free of Bordetella were vaccinated with one of two commercial porcine B. bronchiseptica vaccines, a human DPT vaccine (which includes a Bordetella pertussis component), or an autogenous B. bronchiseptica bacterin. Twenty-one days following vaccination, the animals were challenged with an intranasal dose of 10(6) virulent B. bronchiseptica cells. The animals were euthanized and necropsied 15 days after challenge. The nares, nasopharynx, distal trachea and lungs were cultured. All nonvaccinated control animals developed acute signs of pneumonia, while none of the vaccinated animals developed clinical signs of disease or gross lesions. The frequency of B. bronchiseptica isolation from the lungs of animals in each vaccine group was reduced. However, approximately 70% of all animals in each vaccine group harbored B. bronchiseptica in the trachea, and almost all harbored B bronchiseptica in the nares and nasopharynx. The porcine vaccines appeared to afford protection against acute pulmonary disease in the guinea pig.     

In vivo colonization profile study of Bordetella bronchiseptica in the nasal cavity

Bordetella bronchiseptica chronically infects a wide range of mammals, and resides primarily in the nasal cavity of the infected host. Multiple virulence factors of Bordetella species have been studied in the context of lower respiratory tract infections, but relatively less is known about the bacterial life cycle in the nasal cavity. Evidences were discovered for Bvg intermediate (Bvg(i)) phase expression in vivo and that the major adhesin filamentous hemagglutinin plays a major role in the colonization of B. bronchiseptica in the unciliated olfactory epithelia of the nasal cavity.     

Functional and ultrastructural effects of nontypeable haemophilus influenzae in a hamster trachea organ culture system

Summary A hamster trachea organ culture system was utilized to evaluate quantitatively the effects of a strain of nontypeableHaemophilus influenzae (NTHI) and culture supernatants of the same strain on ciliary activity. Tracheal explants were maintained in organ culture for 96 to 144 h and ciliary activity was observed daily with an inverted microscope. Explants continuously exposed to a strain of NTHI had a progressive decline in ciliary activity which was significantly lower than uninfected controls evaluated concomitantly by 48 h of exposure and thereafter. Histologic studies revealed a progressive degeneration of mucosal cells and exfoliation of ciliated cells. Scanning electron microscopy showed little adherence of NTHI to the mucosal surface. Sterile broth cultures of NTHI and supernatants of organ cultures infected with the same NTHI strain had no adverse effect on ciliary activity. Infected tracheal explants treated with ampicillin 24, 48, or 72 h after continuous bacterial challenge had no significant decline in ciliary activity compared to controls. The lack of adherence and the histologic changes observed when hamster trachea cultures were infected with NTHI suggested a toxin might mediate the damage observed. Broth and organ culture supernatants, however, produced no damage. Therefore, further studies are needed to determine the role, if any, of a toxin in the production of damage to hamster tracheal explants by NTHI. This work was supported by a Merit Review grant from the Veterans Administration and by Grant AI-19641 from the National Institute of Allergy and Infectious Diseases.     

Spider tracheal systems

1. The tracheal structures of spiders belonging to 15 families were investigated. Techniques developed primarily for use with insects were used to visualize spider tracheae. The tracheae were investigated in whole spiders and with serial sections. A macerating agent is described which dissolves the soft-tissues of the spiders without harming the tracheae, or decolourizing the injected dye. 2. A variety of tracheal systems are illustrated using diagrammatic line-drawings and photographs. 3. The variation in the tracheal structures of the spiders investigated in this study is discussed, as well as the use of tracheal structures in spider classification. Spider tracheae are compared with those of insects. 4. A list is given of the major investigations into spider tracheal systems this century.     

Bordetella bronchiseptica modulates macrophage phenotype leading to the inhibition of CD4+ T cell proliferation and the initiation of a Th17 immune response

Bordetella bronchiseptica is a Gram-negative bacterium equipped with several colonization factors that allow it to establish a persistent infection of the murine respiratory tract. Previous studies indicate that B. bronchiseptica adenylate cyclase toxin (ACT) and the type III secretion system (TTSS) synergize to drive dendritic cells into an altered phenotype to down-regulate the host immune response. In this study, we examined the effects of B. bronchiseptica ACT and TTSS on murine bone marrow-derived macrophages. We demonstrate that ACT and TTSS are required for the inhibition of Ag-driven CD4+ T cell proliferation by bacteria-infected macrophages. We identify PGE2 as the mediator of this inhibition, and we show that ACT and the TTSS synergize to increase macrophage production of PGE2. We further demonstrate that B. bronchiseptica can modulate normal macrophage function and drive the immune response toward a Th17 phenotype classified by the significant production of IL-17. In this study, we show that B. bronchiseptica-infected macrophages can induce IL-17 production from naive CD4+ splenocytes, and that lung tissues from B. bronchiseptica-infected mice exhibit a strong Th17 immune response. ACT inhibited surface expression of CD40 and CD86, suppressed TNF-alpha production, and up-regulated IL-6 production. TTSS also synergized with ACT to up-regulate IL-10 and PGE2 secretion. These findings indicate that persistent colonization by B. bronchiseptica may rely on the ability of the bacteria to differentially modulate both macrophage and dendritic cell function leading to an altered adaptive immune response and subsequent bacterial colonization.     

Morphometric analysis of the tracheal walls of the harvestmen Nemastoma lugubre (Arachnida, Opiliones, Nemastomatidae)

The tracheal system of harvestmen consists of two stem tracheae, which give rise to higher order tracheae that supply the extremities and internal organs. In this study, we used stereological morphometric methods to investigate diffusing capacities of the walls ('lateral diffusing capacity') of the tracheae of adult males and females of Nemastoma lugubre. Diffusing barriers of the tracheal walls tend to be thinnest (0.17-0.19 microm) for the smallest tracheae (inner diameter 0.5-2 microm). In other tracheal classes the diffusing barriers increase with increasing diameters. Calculation of the mass-specific diffusing capacity for oxygen (D(O2)) of the walls of all higher order tracheae revealed 10.57 microl min(-1)g(-1)kPa(-1) for the females (mean body mass 3.8 mg) and 25.23 microl min(-1)g(-1)kPa(-1) for the males (mean body mass 1.4 mg). In both animal groups, the main D(O2) (58-67%) lies in the tracheae with an inner diameter of 0.5-2 microm, but also tracheae up to an inner diameter of 20 microm allow gas exchange via the tracheal walls. Stem tracheae are of no importance for lateral diffusion. Our results are consistent with the hypothesis that the functional morphology of the tracheal system of harvestmen represents an 'intermediate state' between the tracheal system of insects in which gas exchange is focused on the distal portions and that of spiders, in which the walls of all tracheae serve in gas exchange.     

Molecular aspects of Bordetella pertussis pathogenesis.

The molecular mechanisms of Bordetella virulence are now well understood, and many virulence factors have been identified and characterized at the molecular level. These virulence factors can be grouped into two major categories: adhesins, such as filamentous hemagglutinin, pertactin and fimbriae, and toxins, such as pertussis toxin, adenylate cyclase, dermonecrotic toxin and tracheal cytotoxin. The production of most virulence factors is coordinately regulated by a two-component signal transduction system composed of the regulator BvgA and the sensor protein BvgS. The adhesins and toxins act in concert to establish infection. Some adhesins exert their effects synergically or are redundant functioning only in the absence of another adhesin, illustrating the importance of adhesion in infection. Most virulence factors are secreted into the culture supernatant or exposed at the surface of the bacterial cell. A notable exception is dermonecrotic toxin, which remains in the cytoplasmic compartment of bacterial cells. Most virulence factors are produced by all of the three major Bordetella species, B. pertussis, B. parapertussis and B. bronchiseptica. However, some, such as pertussis toxin and the tracheal colonization factor, are only produced by B. pertussis. Our understanding of Bordetella virulence at the molecular level has led to the development of new acellular vaccines against whooping cough, and of genetically attenuated B. pertussis strains to be used as recombinant live bacterial vaccine vectors for homologous and heterologous protection.     

Modulation of host immune responses, induction of apoptosis and inhibition of NF-kappaB activation by the Bordetella type III secretion system

Bordetella bronchiseptica establishes respiratory tract infections in laboratory animals with high efficiency. Colonization persists for the life of the animal and infection is usually asymptomatic in immunocompetent hosts. We hypothesize that this reflects a balance between immunostimulatory events associated with infection and immunomodulatory events mediated by the bacteria. We have identified 15 loci that are part of a type III secretion apparatus in B. bronchiseptica and three secreted proteins. The functions of the type III secretion system were investigated by comparing the phenotypes of wild-type bacteria with two strains that are defective in type III secretion using in vivo and in vitro infection models. Type III secretion mutants were defective in long-term colonization of the trachea in immunocompetent mice. The mutants also elicited higher titres of anti- Bordetella antibodies upon infection compared with wild-type bacteria. Type III secretion mutants also showed increased lethal virulence in immunodeficient SCID-beige mice. These observations suggest that type III-secreted products of B. bronchiseptica interact with components of both innate and adaptive immune systems of the host. B. bronchiseptica induced apoptosis in macrophages in vitro and inflammatory cells in vivo and type III secretion was required for this process. Infection of an epithelial cell line with high numbers of wild type, but not type III deficient B. bronchiseptica resulted in rapid aggregation of NF-κB into large complexes in the cytoplasm. NF-κB aggregation was dependent on type III secretion and aggregated NF-κB did not respond to TNFα activation, suggesting B. bronchiseptica may modulate host immunity by inactivating NF-κB. Based on these in vivo and in vitro results, we hypothesize that the Bordetella type III secretion system functions to modulate host immune responses during infection.     

Adherence of ovine and human Bordetella parapertussis to continuous cell lines and ovine tracheal organ culture

The adherence of ovine and human isolates of Bordetella parapertussis to ovine and human continuous culture cell lines and to ovine tracheal organ culture was compared. Adherence to non-ciliated respiratory continuous culture cells did not reveal any host-specificity of the isolates. In contrast, adherence of ovine B. parapertussis strains to ciliated ovine tracheal organ culture was significantly greater than that of human strains. These results indicate that tracheal organ culture is a useful tool for studying host-specific adherence of B. parapertussis and suggest that adherence of B. parapertussis to ciliated epithelia is species-specific making it unlikely that the transfer of B. parapertussis between humans and sheep will result in an infection.     

Association of tracheal placodes with leg primordia in Drosophila and implications for the origin of insect tracheal systems

Adaptation to diverse habitats has prompted the development of distinct organs in different animals to better exploit their living conditions. This is the case for the respiratory organs of arthropods, ranging from tracheae in terrestrial insects to gills in aquatic crustaceans. Although Drosophila tracheal development has been studied extensively, the origin of the tracheal system has been a long-standing mystery. Here, we show that tracheal placodes and leg primordia arise from a common pool of cells in Drosophila, with differences in their fate controlled by the activation state of the wingless signalling pathway. We have also been able to elucidate early events that trigger leg specification and to show that cryptic appendage primordia are associated with the tracheal placodes even in abdominal segments. The association between tracheal and appendage primordia in Drosophila is reminiscent of the association between gills and appendages in crustaceans. This similarity is strengthened by the finding that homologues of tracheal inducer genes are specifically expressed in the gills of crustaceans. We conclude that crustacean gills and insect tracheae share a number of features that raise the possibility of an evolutionary relationship between these structures. We propose an evolutionary scenario that accommodates the available data.     

A role for lipopolysaccharide in turkey tracheal colonization by Bordetella avium as demonstrated in vivo and in vitro

We isolated two insertion mutants of Bordetella avium that exhibited a peculiar clumped-growth phenotype and found them to be attenuated in turkey tracheal colonization. The mutants contained transposon insertions in homologues of the wlbA and wlbL genes of Bordetella pertussis. The wlb genetic locus of B. pertussis has been previously described as containing 12 genes involved in lipopolysaccharide (LPS) biosynthesis. Polyacrylamide gel analysis of LPS from B. avium wlbA and wlbL insertion mutants confirmed an alteration in the LPS profile. Subsequent cloning and complementation of the wlbA and wlbL mutants in trans with a recombinant plasmid containing the homologous wlb locus from B. avium eliminated the clumped-growth phenotype and restored the LPS profile to that of wild-type B. avium. Also, a parental level of tracheal colonization was restored to both mutants by the recombinant plasmid. Interestingly, complementation of the wlbA and wlbL mutants with a recombinant plasmid containing the heterologous wlb locus from B. pertussis, B. bronchiseptica, or Bordetella parapertussis eliminated the clumped-growth phenotype and resulted in a change in the LPS profile, although not to that of wild-type B. avium. The mutants also acquired resistance to a newly identified B. avium-specific bacteriophage, Ba1. Complementation of both wlbA and wlbL mutants with the homologous wlb locus of B. avium, but not the heterologous B. pertussis locus, restored sensitivity to Ba1. Complementation of the wlbL mutant, but not the wlbA mutant, with the heterologous wlb locus of Bordetella bronchiseptica or B. parapertussis restored partial sensitivity to Ba1. Comparisons of the LPS profile and phage sensitivity of the mutants upon complementation by wlb loci from the heterologous species and by B. avium suggested that phage sensitivity required the presence of O-antigen. At the mechanistic level, both mutants showed a dramatic decrease in serum resistance and a decrease in binding to turkey tracheal rings in vitro. In the case of serum resistance, complementation of both mutants with the homologous wlb locus of B. avium restored serum resistance to wild-type levels. However, in the case of epithelial cell binding, only complementation of the wlbA mutant completely restored binding to wild-type levels (binding was only partially restored in the wlbL mutant). This is the first characterization of LPS mutants of B. avium at the genetic level and the first report of virulence changes by both in vivo and in vitro measurements.