Effects of famciclovir in cats with spontaneous acute upper respiratory tract disease.

OBJECTIVES: The aim of this study was to assess the effects of famciclovir administration in cats with spontaneously acquired acute upper respiratory tract disease. METHODS: Twenty-four kittens with clinical signs of acute upper respiratory tract disease were randomly allocated to receive doxycycline (5 mg/kg PO q12h) alone (group D; n = 12) or with famciclovir (90 mg/kg PO q12h; group DF; n = 12) for up to 3 weeks. Clinical disease severity was scored at study entry and daily thereafter. Oculo-oropharyngeal swabs collected at study entry and exit were assessed using quantitative PCR for nucleic acids of feline herpesvirus type 1 (FHV-1), feline calicivirus (FCV), Chlamydia felis, Bordetella bronchiseptica and Mycoplasma felis. RESULTS: The median (range) age of cats was 1.5 (1-6) months in group D vs 1.6 (1-5) months in group DF (P = 0.54). Pathogens detected in oculo-oropharyngeal swabs at study entry included FCV (n = 13/24; 54%), M felis (n = 8/24; 33%), FHV-1 (n = 7/24; 29%), C felis (n = 7/24; 29%) and B bronchiseptica (n = 3/24; 12%). Median (range) duration of clinical signs was 11.5 (3-21) days in group DF and 11 (3-21) days in group D (P = 0.75). Median (range) total disease score at the end of the study did not differ between groups (group D 1 [1-1] vs group DF 1 [1-3]; P = 0.08). CONCLUSIONS AND RELEVANCE: This study revealed no significant difference in response to therapy between cats treated with doxycycline alone or with famciclovir; cats improved rapidly in both groups. However, identification of FHV-1 DNA was relatively uncommon in this study and clinical trials focused on FHV-1-infected cats are warranted to better evaluate famciclovir efficacy.

BteA Secreted from the Bordetella bronchiseptica Type III Secetion System Induces Necrosis through an Actin Cytoskeleton Signaling Pathway and Inhibits Phagocytosis by Macrophages.

BteA is one of the effectors secreted from the Bordetella bronchiseptica type III secretion system. It has been reported that BteA induces necrosis in mammalian cells; however, the roles of BteA during the infection process are largely unknown. In order to investigate the BteA functions, morphological changes of the cells infected with the wild-type B. bronchiseptica were examined by time-lapse microscopy. L2 cells, a rat lung epithelial cell line, spread at 1.6 hours after B. bronchiseptica infection. Membrane ruffles were observed at peripheral parts of infected cells during the cell spreading. BteA-dependent cytotoxicity and cell detachment were inhibited by addition of cytochalasin D, an actin polymerization inhibitor. Domain analyses of BteA suggested that two separate amino acid regions, 200-312 and 400-658, were required for the necrosis induction. In order to examine the intra/intermolecular interactions of BteA, the amino- and the carboxyl-terminal moieties were purified as recombinant proteins from Escherichia coli. The amino-terminal moiety of BteA appeared to interact with the carboxyl-terminal moiety in the pull-down assay in vitro. When we measured the amounts of bacteria phagocytosed by J774A.1, a macrophage-like cell line, the phagocytosed amounts of B. bronchiseptica strains that deliver BteA into the host cell cytoplasm were significantly lower than those of strains that lost the ability to translocate BteA into the host cell cytoplasm. These results suggest that B. bronchiseptica induce necrosis by exploiting the actin polymerization signaling pathway and inhibit macrophage phagocytosis.

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.

Cooperative roles for fimbria and filamentous hemagglutinin in Bordetella adherence and immune modulation.

UNLABELLED: Bordetella fimbriae (FIM) are generally considered to function as adhesins despite a lack of experimental evidence supporting this conclusion for Bordetella pertussis and evidence against a requirement for FIM in adherence of Bordetella bronchiseptica to mammalian cell lines. Using B. bronchiseptica and mice, we developed an in vivo adherence assay that revealed that FIM do function as critically important adhesins in the lower respiratory tract. In the first few days postinoculation, FIM-deficient B. bronchiseptica induced a more robust inflammatory response than wild-type bacteria did, suggesting that FIM, like filamentous hemagglutinin (FHA), allow B. bronchiseptica to suppress the innate immune response to infection. Localization analyses indicated that FIM are required for efficient attachment to airway epithelium, as bacteria lacking FIM localized to alveoli. FHA-deficient bacteria, in contrast, localized to airways. Bacteria unable to produce both FIM and FHA localized to alveoli and caused increased inflammation and histopathology identical to that caused by FIM-deficient bacteria, demonstrating that lack of FIM is epistatic to lack of FHA. Coinoculation experiments provided evidence that wild-type B. bronchiseptica suppresses inflammation locally within the respiratory tract and that both FHA and FIM are required for defense against clearance by the innate immune system. Altogether, our data suggest that FIM-mediated adherence to airway epithelium is a critical first step in Bordetella infection that allows FHA-dependent interactions to mediate tight adherence, suppression of inflammation, and resistance to inflammatory cell-mediated clearance. Our results suggest that mucosal antibodies capable of blocking FIM-mediated interactions could prevent bacterial colonization of the lower respiratory tract. IMPORTANCE: Although fimbriae (FIM) have been shown to be important mediators of adherence for many bacterial pathogens, there is surprisingly little experimental evidence supporting this role for Bordetella fimbria. Our results provide the first demonstration that Bordetella FIM function as adhesins in vivo, specifically to airway epithelium. Furthermore, our results suggest that FIM mediate initial interactions with airway epithelial cells that are followed by tight filamentous hemagglutinin (FHA)-mediated binding and that together, FIM and FHA allow Bordetella to suppress inflammation, leading to prolonged colonization. Given the shortcoming of the current acellular component pertussis (aP) vaccine in preventing colonization, these findings suggest that generation of antibodies capable of blocking FIM-mediated adherence could potentially prevent Bordetella colonization.

A Type VI secretion system encoding locus is required for Bordetella bronchiseptica immunomodulation and persistence in vivo.

Type VI Secretion Systems (T6SSs) have been identified in numerous gram-negative pathogens, but the lack of a natural host infection model has limited analysis of T6SS contributions to infection and pathogenesis. Here, we describe disruption of a gene within locus encoding a putative T6SS in Bordetella bronchiseptica strain RB50, a respiratory pathogen that circulates in a broad range of mammals, including humans, domestic animals, and mice. The 26 gene locus encoding the B. bronchiseptica T6SS contains apparent orthologs to all known core genes and possesses thirteen novel genes. By generating an in frame deletion of clpV, which encodes a putative ATPase required for some T6SS-dependent protein secretion, we observe that ClpV contributes to in vitro macrophage cytotoxicity while inducing several eukaryotic proteins associated with apoptosis. Additionally, ClpV is required for induction of IL-1ß, IL-6, IL-17, and IL-10 production in J774 macrophages infected with RB50. During infections in wild type mice, we determined that ClpV contributes to altered cytokine production, increased pathology, delayed lower respiratory tract clearance, and long term nasal cavity persistence. Together, these results reveal a natural host infection system in which to interrogate T6SS contributions to immunomodulation and pathogenesis.

sigE facilitates the adaptation of Bordetella bronchiseptica to stress conditions and lethal infection in immunocompromised mice.

BACKGROUND: The cell envelope of a bacterial pathogen can be damaged by harsh conditions in the environment outside a host and by immune factors during infection. Cell envelope stress responses preserve the integrity of this essential compartment and are often required for virulence. Bordetella species are important respiratory pathogens that possess a large number of putative transcription factors. However, no cell envelope stress responses have been described in these species. Among the putative Bordetella transcription factors are a number of genes belonging to the extracytoplasmic function (ECF) group of alternative sigma factors, some of which are known to mediate cell envelope stress responses in other bacteria. Here we investigate the role of one such gene, sigE, in stress survival and pathogenesis of Bordetella bronchiseptica. RESULTS: We demonstrate that sigE encodes a functional sigma factor that mediates a cell envelope stress response. Mutants of B. bronchiseptica strain RB50 lacking sigE are more sensitive to high temperature, ethanol, and perturbation of the envelope by SDS-EDTA and certain ß-lactam antibiotics. Using a series of immunocompromised mice deficient in different components of the innate and adaptive immune responses, we show that SigE plays an important role in evading the innate immune response during lethal infections of mice lacking B cells and T cells. SigE is not required, however, for colonization of the respiratory tract of immunocompetent mice. The sigE mutant is more efficiently phagocytosed and killed by peripheral blood polymorphonuclear leukocytes (PMNs) than RB50, and exhibits decreased cytotoxicity toward macrophages. These altered interactions with phagocytes could contribute to the defects observed during lethal infection. CONCLUSIONS: Much of the work on transcriptional regulation during infection in B. bronchiseptica has focused on the BvgAS two-component system. This study reveals that the SigE regulon also mediates a discrete subset of functions associated with virulence. SigE is the first cell envelope stress-sensing system to be described in the bordetellae. In addition to its role during lethal infection of mice deficient in adaptive immunity, our results indicate that SigE is likely to be important for survival in the face of stresses encountered in the environment between hosts.

Microarray and functional analysis of growth phase-dependent gene regulation in Bordetella bronchiseptica.

Growth phase-dependent gene regulation has recently been demonstrated to occur in Bordetella pertussis, with many transcripts, including known virulence factors, significantly decreasing during the transition from logarithmic to stationary-phase growth. Given that B. pertussis is thought to have derived from a Bordetella bronchiseptica-like ancestor, we hypothesized that growth phase-dependent gene regulation would also occur in B. bronchiseptica. Microarray analysis revealed and quantitative real-time PCR (qRT-PCR) confirmed that growth phase-dependent gene regulation occurs in B. bronchiseptica, resulting in prominent temporal shifts in global gene expression. Two virulence phenotypes associated with these gene expression changes were tested. We found that growth-dependent increases in expression of some type III secretion system (TTSS) genes led to a growth phase-dependent increase in a TTSS-dependent function, cytotoxicity. Although the transcription of genes encoding adhesins previously shown to mediate adherence was decreased in late-log and stationary phases, we found that the adherence of B. bronchiseptica did not decrease in these later phases of growth. Microarray analysis revealed and qRT-PCR confirmed that growth phase-dependent gene regulation occurred in both Bvg(+) and Bvg(-) phase-locked mutants, indicating that growth phase-dependent gene regulation in B. bronchiseptica can function independently from the BvgAS regulatory system.

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.

Functional BvgAS virulence control system in Bordetella bronchiseptica is necessary for induction of Ca2+ transients in ciliated tracheal epithelial cells.

To study initial Bordetella bronchiseptica-tracheal epithelial cell interactions, we coincubated B. bronchiseptica with rabbit tracheal explant cultures and assayed bacterial adherence and host cell Ca(2+) signaling. Wild-type B. bronchiseptica (RB50) preferentially adhered to cilia and induced ciliated host cell Ca(2+) transients within 2 min of coincubation, whereas coincubation with an avirulent strain (RB57) resulted in limited binding and Ca(2+) signaling. The described cell system allows for assessment of initial B. bronchiseptica-host cell interactions that can contribute to pathogenicity or to host cell defense.

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.

Phagosome acidification has opposite effects on intracellular survival of Bordetella pertussis and B. bronchiseptica.

Bordetella pertussis is readily killed after uptake by professional phagocytes, whereas its close relative Bordetella bronchiseptica is not and can persist intracellularly for days. Phagocytosis of members of either species by a mouse macrophage cell line results in transport of the bacteria to a phagosomal compartment positive for the lysosome-associated membrane protein 1, the protease cathepsin D, and the late endosomal vacuolar proton-pumping ATPase but negative for the early endosome antigen 1 and the early endosomal transferrin receptor. In addition, we demonstrate that Bordetella-containing phagosomes rapidly acidify to pH 4.5 to 5.0. Taken together, these data demonstrate that Bordetella-containing phagosomes rapidly mature to an acidic late endosomal/lysosomal compartment. Following up on this observation, we determined that B. pertussis does not survive in bacterial growth media adjusted to a pH of 4.5, whereas this pH has only minor effects on the growth of B. bronchiseptica. Raising the intracellular pH in infected macrophages by the addition of bafilomycin A(1), ammonium chloride, or monensin increases the survival of acid-sensitive B. pertussis but, surprisingly, decreases that of acid-tolerant B. bronchiseptica. In summary, we hypothesize that the differential survival of B. pertussis and B. bronchiseptica in macrophages is, at least in part, due to the differences in their acid tolerance.

Role of Bordetella bronchiseptica fimbriae in tracheal colonization and development of a humoral immune response.

Fimbriae are filamentous, cell surface structures which have been proposed to mediate attachment of Bordetella species to respiratory epithelium. Bordetella bronchiseptica has four known fimbrial genes: fim2, fim3, fimX, and fimA. While these genes are unlinked on the chromosome, their protein products are assembled and secreted by a single apparatus encoded by the fimBCD locus. The fimBCD locus is embedded within the fha operon, whose genes encode another putative adhesin, filamentous hemagglutinin (FHA). We have constructed a Fim(-) B. bronchiseptica strain, RB63, by introducing an in-frame deletion extending from fimB through fimD. Western blot analysis showed that RB63 is unable to synthesize fimbriae but is unaffected for FHA expression. Using this mutant, we assessed the role of fimbriae in pathogenesis in vitro and in vivo in natural animal hosts. Although RB63 was not significantly defective in its ability to adhere to various tissue culture cell lines, including human laryngeal HEp-2 cells, it was considerably altered in its ability to cause respiratory tract infections in rats. The number of DeltafimBCD bacteria recovered from the rat trachea at 10 days postinoculation was significantly decreased compared to that of wild-type B. bronchiseptica and was below the limit of detection at 30 and 60 days postinoculation. The number of bacteria recovered from the nasal cavity and larynx was not significantly different between RB63 and the wild-type strain at any time point. The ability of fimbriae to mediate initial attachment to tracheal tissue was tested in an intratracheal inoculation assay. Significantly fewer RB63 than wild-type bacteria were recovered from the tracheas at 24 h after intratracheal inoculation. These results demonstrate that fimbriae are involved in enhancing the ability of B. bronchiseptica to establish tracheal colonization and are essential for persistent colonization at this site. Interestingly, anti-Bordetella serum immunoglobulin M (IgM) levels were significantly lower in animals infected with RB63 than in animals infected with wild-type B. bronchiseptica at 10 days postinoculation. Even at 30 days postinoculation, RB63-infected animals had lower serum anti-Bordetella antibody titers in general. This disparity in antibody profiles suggests that fimbriae are also important for the induction of a humoral immune response.

Non-opsonic attachment of Bordetella bronchiseptica mediated by CD11/CD18 and cell surface carbohydrates.

Porcine atrophic rhinitis associated with Bordetella bronchiseptica is characterized by a severe inflammatory response in the mucosa of the nasal turbinates. Initial infiltrates of polymorphonuclear leukocytes (PMN) are followed by accumulations of mononuclear cells. In this report, we have investigated the interaction between porcine PMN and B. bronchiseptica. PMN incubated in PBS with a fluorescently labeled hemagglutinating porcine isolate, but not a non-hemagglutinating variant, had high levels of cell-associated fluorescence as determined by flow cytometry. Light microscopy indicated that most cell-associated bacteria were ingested. Transmission electron microscopy confirmed the presence of intracellular bacteria, which were contained within membrane-bound phagosomes. A monoclonal antibody specific for the leukocyte integrin polypeptide CD18 partially inhibited attachment of B. bronchiseptica to normal PMN but not to PMN genetically deficient in CD11/CD18 integrins. Higher levels of inhibition occurred when bacteria and normal PMN were co-incubated in the presence of galactose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, mannose and methyl alpha-D-mannopyranoside. D-glucose, L-fucose, alpha-lactose and sialic acid had no inhibitory effect. We conclude that B. bronchiseptica is readily ingested by porcine PMN in the absence of complement and antibody and that internalization is mediated by multiple adhesion mechanisms, including CD18- and carbohydrate-dependent ones.