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Traditionally, eosinophils have been linked to parasitic infections and pathological disease states. However, emerging literature has unveiled a more nuanced and intricate role for these cells, demonstrating their key functions in maintaining mucosal homeostasis. Eosinophils exhibit diverse phenotypes and exert multifaceted effects during infections, ranging from promoting pathogen persistence to triggering allergic reactions. Our investigations primarily focus on Bordetella spp., with particular emphasis on Bordetella bronchiseptica , a natural murine pathogen that induces diseases in mice akin to pertussis in humans. Recent findings from our published work have unveiled a striking interaction between B. bronchiseptica and eosinophils, facilitated by the btrS -mediated mechanism. This interaction serves to enhance pathogen persistence while concurrently delaying adaptive immune responses. Notably, this role of eosinophils is only noted in the absence of a functional btrS signaling pathway, indicating that wild-type B. bronchiseptica , and possibly other Bordetella spp., possess such adeptness in manipulating eosinophils that the true function of these cells remains obscured during infection. In this review, we present the mounting evidence pointing toward eosinophils as targets of bacterial exploitation, facilitating pathogen persistence and fostering chronic infections in diverse mucosal sites, including the lungs, gut, and skin. We underscore the pivotal role of the master regulator of Bordetella pathogenesis, the sigma factor BtrS, in orchestrating eosinophil-dependent immunomodulation within the context of pulmonary infection. These putative convergent strategies of targeting eosinophils offer promising avenues for the development of novel therapeutics targeting respiratory and other mucosal pathogens., Competing Interests: The authors declare no conflict of interest.

Bordetella bronchiseptica is a pathogen causing respiratory infections in mammals. With the improving understanding of companion animals' welfare, addressing the side effects of bordetella vaccine gains importance in dogs. Studies on diverse subunit vaccines are actively pursued in humans to safely and effectively control bordetellosis. Therefore, our objective was to develop a canine bordetella vaccine inspired by human vaccine development. We evaluated the immunogenicity of the two bacterial components: the outer membrane proteins (OMPs) and the dermonecrotic toxin (DNT) from a canine isolate of B. bronchiseptica. In-silico analysis identified eight domains of DNT, and Domain 3 was selected as the most promising antigen candidate. Additionally, the OMPs were extracted and examined using SDS-PAGE and Western blot analysis. The distinct immunological characteristic of OMPs and DNT-3 were examined individually and in combination. Gene expression and cytokine production were also evaluated in DH82 cells after stimulation with those antigens. Treatment with OMPs resulted in higher level of Th1 related cytokines, while DNT-3 induced a predominant response associated with Th17 and Th2 in the cytokine production. Synergistic effects were observed exclusively on IL-23, indicating increase of a potential risk of side effects when OMPs and DNT act together. These findings provide valuable insights into the reactogenicity of conventional Bordetella vaccines. Further, the presented preclinical data in this study offer an alternative method of the development for an optimal next-generation Bordetella vaccine for companion animals and humans, replacing the acellular vaccines containing both toxin and protein components., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Swine atrophic rhinitis is a disease caused by Pasteurella multocida and Bordetella bronchiseptica that affects pigs. Inactivated vaccines containing the toxins produced by Pasteurella multocida and Bordetella bronchiseptica have been widely used for the prevention of swine atrophic rhinitis. The efficacy of a vaccine is correlated with the amount of antigen present; however, the protective toxin of P. multocida bound to aluminum hydroxide, which is used as an adjuvant, can hinder the monitoring of the antigen concentration in the vaccine. This study assessed the applicability of a dot immunoassay as an antigen quantification method using monoclonal antibodies. This quantification method was able to detect the antigen with high specificity and sensitivity even when the antigen was bound to the adjuvant, and its application to vaccine products revealed a correlation between the amount of antigen present in the vaccine and the neutralizing antibody titers induced in pigs. The antigen quantification method presented in this study is a simple and sensitive assay capable of quantifying the amount of antigen present in a vaccine that can be used as an alternative quality control measure., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Yoshimura et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

What Is Known and Objective: The Gram-negative bacterium, Bordetella bronchiseptica, causes lower airway respiratory disease in people with cystic fibrosis (CF), as well as in companion animals, especially dogs. Presently, there are several acellular vaccines available for B. pertussis but no vaccine available for B. bronchiseptica. However given the shared protein homology between these two closely related species, we wished to explore whether pertussis vaccines may offer some cross-protection against B. bronchiseptica., Comment: Bordetella pertussis and B. bronchiseptica are closely related phylogenetically, as well as sharing protein homology in several pertussis vaccine components, including (i) pertussis toxin (PT), (ii) filamentous haemagglutinin (FHA), (iii) pertactin and (iv) fimbriae (types 2 and 3). Given that pertussis vaccine contains cross-reactive antigens with B. bronchiseptica, licensed pertussis vaccines may therefore offer cross-protection against B. bronchiseptica., What Is New and Conclusion: Cystic fibrosis pet owners should ensure that they have an up-to-date vaccination record relating to their pertussis vaccine. Although no monovalent human pertussis vaccines are currently available, licensed non-live booster vaccines for B. pertussis are available for individuals in the age range >10 years old. People with CF should ensure that they are adequately and currently protected against pertussis, to avoid whooping cough, which may also offer some cross-protection against B. bronchiseptica and therefore help further mitigate the risk of zoonotic infection of this organism from pets to their owners., (© 2021 John Wiley & Sons Ltd.)

Eosinophils are granulocytes primarily associated with T H 2 responses to parasites or immune hyper-reactive states, such as asthma, allergies, or eosinophilic esophagitis. However, it does not make sense from an evolutionary standpoint to maintain a cell type that is only specific for parasitic infections and that otherwise is somehow harmful to the host. In recent years, there has been a shift in the perception of these cells. Eosinophils have recently been recognized as regulators of immune homeostasis and suppressors of over-reactive pro-inflammatory responses by secreting specific molecules that dampen the immune response. Their role during parasitic infections has been well investigated, and their versatility during immune responses to helminths includes antigen presentation as well as modulation of T cell responses. Although it is known that eosinophils can present antigens during viral infections, there are still many mechanistic aspects of the involvement of eosinophils during viral infections that remain to be elucidated. However, are eosinophils able to respond to bacterial infections? Recent literature indicates that Helicobacter pylori triggers T H 2 responses mediated by eosinophils; this promotes anti-inflammatory responses that might be involved in the long-term persistent infection caused by this pathogen. Apparently and on the contrary, in the respiratory tract, eosinophils promote T H 17 pro-inflammatory responses during Bordetella bronchiseptica infection, and they are, in fact, critical for early clearance of bacteria from the respiratory tract. However, eosinophils are also intertwined with microbiota, and up to now, it is not clear if microbiota regulates eosinophils or vice versa, or how this connection influences immune responses. In this review, we highlight the current knowledge of eosinophils as regulators of pro and anti-inflammatory responses in the context of both infection and naïve conditions. We propose questions and future directions that might open novel research avenues in the future.

Classical Bordetella species are primarily isolated from animals and humans causing asymptomatic infection to lethal pneumonia. However, isolation of these bacteria from any extra-host environmental niche has not been reported so far. Here, we have characterized the genomic plasticity and antibody response of Bordetella bronchiseptica strain HT200, isolated from a thermal spring. Genomic ANI value and SNPs-based phylogenetic tree suggest a divergent evolution of strain HT200 from a human-adapted lineage of B. bronchiseptica. Growth and survivability assay showed strain HT200 retained viability for more than 5 weeks in the filter-sterilized spring water. In addition, genes or loci encoding the Bordetella virulence factors such as DNT, ACT and LPS O-antigen were absent in strain HT200, while genes encoding other virulence factors were highly divergent. Phenotypically, strain HT200 was non-hemolytic and showed weak hemagglutination activity, but was able to colonize in the respiratory organs of mice. Further, both infection and vaccination with strain HT200 induced protective antibody response in mouse against challenge infection with virulent B. bronchiseptica strain RB50. In addition, genome of strain HT200 (DSM 26023) showed presence of accessory genes and operons encoding predicted metabolic functions pertinent to the ecological conditions of the thermal spring., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

Vaccines, in many cases, stimulate only too weak immunogenicity to prevent infection. Therefore, adjuvants are required during their preparation to boost the immune response. We herein developed a PEGylated nano-adjuvant based on Rehmannia glutinosa polysaccharide (RGP). The addition of PEG layer exhibits enhanced immune performance of the nano-RGP. Stimulation of dendritic cells (DCs) with PEGylated nano-RGP (pRL) led to increased proliferation and cytokine production (IL-6, IL-12, IL-1β and TNF-α). The pRL was internalized into DCs via a rapid and efficient method. The mice immunized with pRL exhibited enhanced antigen-specific serum IgG and Th1-(IFN-γ), Th2-(IL-4), and Th17-(IL-17, IL-6) cytokine production, contributing to a good anti-infection performance. Furthermore, the pRL could effectively deliver the antigen to the lymph nodes (LNs), activate DC in the LN and produce enhanced CD4 + and CD8 + T-cells-derived memory (CD44 high CD62L high ), and effector (CD44 high CD62L low ) as well as functional phenotypes. Our results revealed that pRL can act as a promising adjuvant with targeted delivery of antigen due to its effective activation and robust adaptive immunity induction of DCs., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Bordetella bronchiseptica ( B . bronchiseptica ) is an obligately aerobic, oxidase- and catalase-positive, nonfermentative Gram-negative coccobacillus. This study is aimed at examining the immune effects of Rg1, Rg1 plus oil, and other common adjuvants on inactivated B . bronchiseptica vaccine in rabbits. The mechanism underlying the adjuvant effect of Rg1 plus oil on the vaccine was also explored. Rg1 (100  μ g) plus oil significantly improved the immune effect of B . bronchiseptica vaccine at both the humoral and cellular levels. Rg1-oil adjuvant increased the levels of IL-2 and IL-4 in rabbits after immunization. Rg1 (100  μ g) plus oil also significantly increased TLR2 expression and downregulated NF- κ B in splenocytes. Rg1-oil adjuvant may increase the levels of IL-2 and IL-4 via upregulating TLR2, thereby enhancing the immune effect of B . bronchiseptica vaccine. In conclusion, Rg1 plus oil could be used as a potential vaccine adjuvant for rabbit B . bronchiseptica vaccine., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Xiao Chenwen et al.)

Temperate phages are considered as natural vectors for gene transmission among bacteria due to the ability to integrate their genomes into a host chromosome, therefore, affect the fitness and phenotype of host bacteria. Many virulence genes of pathogenic bacteria were identified in temperate phage genomes, supporting the concept that temperate phages play important roles in increasing the bacterial pathogenicity through delivery of the virulence genes. However, little is known about the roles of temperate phages in attenuation of bacterial virulence. Here, we report a novel Bordetella bronchiseptica temperate phage, vB_BbrS_PHB09 (PHB09), which has a 42,129-bp dsDNA genome with a G+C content of 62.8%. Phylogenetic analysis based on large terminase subunit indicated that phage PHB09 represented a new member of the family Siphoviridae. The genome of PHB09 contains genes encoding lysogen-associated proteins, including integrase and cI protein. The integration site of PHB09 is specifically located within a pilin gene of B. bronchiseptica . Importantly, we found that the integration of phage PHB09 significantly decreased the virulence of parental strain B. bronchiseptica Bb01 in mice, most likely through disruption the expression of pilin gene. Moreover, a single shot of the prophage bearing B. bronchiseptica strain completely protected mice against lethal challenge with wild-type virulent B. bronchiseptica , indicating the vaccine potential of lysogenized strain. Our findings not only indicate the complicated roles of temperate phages in bacterial virulence other than simple delivery of virulent genes but also provide a potential strategy for developing bacterial vaccines., (Copyright © 2020 Chen, Yang, Yang, Song, Wang, Sun, Gu, Chen, Tong, Tao and Wu.)

Aims: The outer membrane porin protein (OMPP) of Bordetella bronchiseptica is an important adhesion factor and protective immunogen. The aim of this study was to verify the immunogenicity of recombinant OMPP and its protective efficacy against a lethal challenge with B. bronchiseptica in rabbits., Methods and Results: Soluble rOMPP was successfully expressed in Escherichia coli, and the purified recombinant protein was mixed with the ISA 201 VG adjuvant to prepare a subunit vaccine for B. bronchiseptica. Rabbits were immunized with the rOMPP subunit vaccine and then infected with the virulent B. bronchiseptica strain QDBb01. Rabbits immunized with the subunit vaccine were completely protected compared to the control group, and the protective effect was obviously better than that of the inactivated whole-cell vaccine. Moreover, analysis of the immunization duration showed that the rOMPP subunit vaccine provided immune protection for at least 4 months after the second immunization., Conclusions: The rOMPP subunit vaccine completely protected rabbits from a subsequent B. bronchiseptica challenge., Significance and Impact of the Study: The results will provide key information for the development of a safe and effective recombinant subunit vaccine against B. bronchiseptica in rabbits., (© 2019 The Society for Applied Microbiology.)

Bordetella bronchiseptica is an etiologic agent of respiratory diseases in animals and humans. Despite the widespread use of veterinary B. bronchiseptica vaccines, there is limited information on their composition and relative efficacy and on the immune responses that they elicit. Furthermore, human B. bronchiseptica vaccines are not available. We leveraged the dual antigenic and adjuvant functions of Bordetella colonization factor A (BcfA) to develop acellular B. bronchiseptica vaccines in the absence of an additional adjuvant. BALB/c mice immunized with BcfA alone or a trivalent vaccine containing BcfA and the Bordetella antigens FHA and Prn were equally protected against challenge with a prototype B. bronchiseptica strain. The trivalent vaccine protected mice significantly better than the canine vaccine Bronchicine and provided protection against a B. bronchiseptica strain isolated from a dog with kennel cough. Th1/17-polarized immune responses correlate with long-lasting protection against bordetellae and other respiratory pathogens. Notably, BcfA strongly attenuated the Th2 responses elicited by FHA and Prn, resulting in Th1/17-skewed responses in inherently Th2-skewed BALB/c mice. Thus, BcfA functions as both an antigen and an adjuvant, providing protection as a single-component vaccine. BcfA-adjuvanted vaccines may improve the efficacy and durability of vaccines against bordetellae and other pathogens., (Copyright © 2019 American Society for Microbiology.)

The Bordetella species are Gram-negative bacterial pathogens that colonizes mammalian respiratory tract causing respiratory diseases in humans and animals. B. bronchiseptica causes clinical conditions in many mammals including immunocompromised humans. Using the dog model of respiratory infection, it has been shown in this study that a newly developed B. bronchiseptica Bacterial Ghost (BbBG) vaccine exhibited significant protection in the face of a severe pathogenic bacterial challenge in seronegative dogs. The protein E -specific lysis mechanism was used to produce BbBGs. Bacterial Ghosts (BGs) are the empty cell envelope of Gram-negative bacterium. They are genetically processed to form a microscopic hole in their membrane, through which all the cytoplasmic contents are expelled leaving behind intact empty bacterial shells. Due to the intact surface structures of BGs, they offer the safety of inactivated but efficacy of live attenuated vaccines. In this study, seronegative dogs were vaccinated subcutaneously (s/c) with two different doses of a newly developed BbBG vaccine [lower 10 ∧ 5 (BbBG - 5) and higher 10 ∧ 7 (BbBG - 7)] on day 0 and 21. The animals were challenged (by aerosol) with virulent live B. bronchiseptica strains 41 days after first vaccination. The dogs vaccinated s/c with BbBG - 7 vaccine had significantly lower spontaneous coughing scores ( P = 0.0001) than dogs in negative control group. Furthermore, the tested BbBG - 7 vaccine was equivalent to the positive control vaccine Bronchicine CAe in terms of safety and efficacy. For the first time, we report the successful use of liquid formulated BGs vaccines in animal studies. Earlier reported studies using BGs vaccines were performed with resuspended freeze-dried BGs preparations.

Intranasally administered live-attenuated influenza virus (LAIV) vaccines provide significant protection against heterologous influenza A virus (IAV) challenge. However, LAIV administration can modify the bacterial microbiota in the upper respiratory tract, including alterations in species that cause pneumonia. We sought to evaluate the effect of Bordetella bronchiseptica colonization on LAIV immunogenicity and efficacy in swine, and the impact of LAIV and IAV challenge on B. bronchiseptica colonization and disease. LAIV immunogenicity was not significantly impacted by B. bronchiseptica colonization, but protective efficacy against heterologous IAV challenge in the upper respiratory tract was impaired. Titers of IAV in the nose and trachea of pigs that received LAIV were significantly reduced when compared to non-vaccinated, challenged controls, regardless of B. bronchiseptica infection. Pneumonia scores were higher in pigs colonized with B. bronchiseptica and challenged with IAV, but this was regardless of LAIV vaccination status. While LAIV vaccination provided significant protection against heterologous IAV challenge, the protection was not sterilizing and IAV replicated in the respiratory tract of all LAIV vaccinated pig. The interaction between IAV, B. bronchiseptica , and host led to development of acute-type B. bronchiseptica lesions in the lung. Thus, the data presented do not negate the efficacy of LAIV vaccination, but instead indicate that controlling B. bronchiseptica colonization in swine could limit the negative interaction between IAV and Bordetella on swine health.

The respiratory tract is constantly exposed to the environment and displays a favorable niche for colonizing microorganisms. However, the effects of respiratory bacterial carriage on the immune system and its implications for secondary responses remain largely unclear. We have employed respiratory carriage with Bordetella bronchiseptica as the underlying model to comprehensively address effects on subsequent immune responses. Carriage was associated with the stimulation of Bordetella -specific CD4⁺, CD8⁺, and CD4⁺CD25⁺Foxp3⁺ T cell responses, and broad transcriptional activation was observed in CD4⁺CD25⁺ T cells. Importantly, transfer of leukocytes from carriers to acutely B. bronchiseptica infected mice, resulted in a significantly increased bacterial burden in the recipient's upper respiratory tract. In contrast, we found that respiratory B. bronchiseptica carriage resulted in a significant benefit for the host in systemic infection with Listeria monocytogenes. Adaptive responses to vaccination and influenza A virus infection, were unaffected by B. bronchiseptica carriage. These data showed that there were significant immune modulatory processes triggered by B. bronchiseptica carriage, that differentially affect subsequent immune responses. Therefore, our results demonstrated the complexity of immune regulation induced by respiratory bacterial carriage, which can be beneficial or detrimental to the host, depending on the pathogen and the considered compartment.

Bordetella bronchiseptica , a Gram-negative bacterium, causes chronic respiratory tract infections in a wide variety of mammalian hosts, including humans (albeit rarely). We recently designed Bordetella pertussis and Bordetella parapertussis experimental vaccines based on outer membrane vesicles (OMVs) derived from each pathogen, and we obtained protection against the respective infections in mice. Here, we demonstrated that OMVs derived from virulent-phase B. bronchiseptica (OMVBbvir + ) protected mice against sublethal infections with different B. bronchiseptica strains, two isolated from farm animals and one isolated from a human patient. In all infections, we observed that the B. bronchiseptica loads were significantly reduced in the lungs of vaccinated animals; the lung-recovered CFU were decreased by ≥4 log units, compared with those detected in the lungs of nonimmunized animals ( P < 0.001). In the OMVBbvir + -immunized mice, we detected IgG antibody titers against B. bronchiseptica whole-cell lysates, along with an immune serum having bacterial killing activity that both recognized B. bronchiseptica lipopolysaccharides and polypeptides such as GroEL and outer membrane protein C (OMPc) and demonstrated an essential protective capacity against B. bronchiseptica infection, as detected by passive in vivo transfer experiments. Stimulation of cultured splenocytes from immunized mice with OMVBbvir + resulted in interleukin 5 (IL-5), gamma interferon (IFN-γ), and IL-17 production, indicating that the vesicles induced mixed Th2, Th1, and Th17 T-cell immune responses. We detected, by adoptive transfer assays, that spleen cells from OMVBbvir + -immunized mice also contributed to the observed protection against B. bronchiseptica infection. OMVs from avirulent-phase B. bronchiseptica and the resulting induced immune sera were also able to protect mice against B. bronchiseptica infection. IMPORTANCE Bordetella bronchiseptica , a Gram-negative bacterium, causes chronic respiratory tract infections in a wide variety of mammalian hosts, including humans (albeit rarely). Several vaccines aimed at preventing B. bronchiseptica infection have been developed and used, but a safe effective vaccine is still needed. The significance and relevance of our research lie in the characterization of the OMVs derived from B. bronchiseptica as the source of a new experimental vaccine. We demonstrated here that our formulation based on OMVs derived from virulent-phase B. bronchiseptica (OMVBbvir + ) was effective against infections caused by B. bronchiseptica isolates obtained from different hosts (farm animals and a human patient). In vitro and in vivo characterization of humoral and cellular immune responses induced by the OMVBbvir + vaccine enabled a better understanding of the mechanism of protection necessary to control B. bronchiseptica infection. Here we also demonstrated that OMVs derived from B. bronchiseptica in the avirulent phase and the corresponding induced humoral immune response were able to protect mice from B. bronchiseptica infection. This realization provides the basis for the development of novel vaccines not only against the acute stages of the disease but also against stages of the disease or the infectious cycle in which avirulence factors could play a role., (Copyright © 2018 American Society for Microbiology.)

We previously demonstrated that Bordetella ( B .) bronchiseptica antigen (Ag) showed high immunostimulatory effects on mouse bone marrow cells (BMs) while Mycoplasma ( M .) hyopneumoniae Ag showed low effects. The focus of this study was to determine if B. bronchiseptica Ag can enhance the M. hyopneumoniae Ag-specific immune response and whether the host's immune system can recognize both Ags. MTT assay results revealed that each or both Ags did not significantly change BM metabolic activity. Flow cytometry analysis using carboxyfluorescein succinimidyl ester showed that B. bronchiseptica Ag can promote the division of BMs. In cytokine and nitric oxide (NO) assays, B. bronchiseptica Ag boosted production of tumor necrosis factor-alpha in M. hyopneumoniae Ag-treated BMs, and combined treatment with both Ags elevated the level of NO in BMs compared to that from treatment of M. hyopneumoniae Ag alone. Immunoglobulin (Ig)G enzyme-linked immunosorbent assay using the sera of Ag-injected mice clearly indicated that B. bronchiseptica Ag can increase the production of M. hyopneumoniae Ag-specific IgG. This study provided information valuable in the development of M. hyopneumoniae vaccines and showed that B. bronchiseptica Ag can be used both as a vaccine adjuvant and as a vaccine Ag.

In order to determine the comparative efficacy of injectable and intranasal vaccines to stimulate Bordetella bronchiseptica (Bb) -reactive anamnestic antibodies, a trial was conducted using 144 adult household dogs of various breeds and ages, which had been previously administered intranasal Bb vaccine approximately 12 months before enrollment. Dogs were randomized into 2 groups and blood, nasal swabs, and pharyngeal swabs were collected prior to the administration of single component Bb vaccines intranasally or parenterally. Ten to 14 days later all dogs were resampled to measure changes in systemic and local antibody to Bb. There were no differences in the changes in Bb -reactive serum IgG and nasal IgA between the groups, whereas intranasally vaccinated dogs had significantly higher Bb -reactive serum IgA. These data indicate that both of the current generation of intranasal (modified-live) and injectable (acellular) Bb vaccines can stimulate anamnestic local and systemic antibody responses in previously vaccinated, Bb -seropositive adult household dogs., Competing Interests: Conflict of interest statement One of the authors (EB) is an employee of Zoetis. Zoetis played no direct role in the acquisition of data, or in the analysis of data. None of the authors has any other financial or personal relationships that could inappropriately influence or bias the content of the paper.

Bordetella bronchiseptica is pervasive in swine populations and plays multiple roles in respiratory disease. Additionally, B. bronchiseptica is capable of establishing long-term or chronic infections in swine. Bacterial biofilms are increasingly recognized as important contributors to chronic bacterial infections. Recently the polysaccharide locus bpsABCD has been demonstrated to serve a critical role in the development of mature biofilms formed by the sequenced laboratory strain of B. bronchiseptica We hypothesized that swine isolates would also have the ability to form mature biofilms and the bpsABCD locus would serve a key role in this process. A mutant containing an in-frame deletion of the bpsABCD structural genes was constructed in a wild-type swine isolate and found to be negative for poly-N-acetylglucosamine (PNAG)-like material by immunoblot assay. Further, the bpsABCD locus was found to be required for the development and maintenance of the three-dimensional structures under continuous-flow conditions. To investigate the contribution of the bpsABCD locus to the pathogenesis of B. bronchiseptica in swine, the KM22Δ bps mutant was compared to the wild-type swine isolate for the ability to colonize and cause disease in pigs. The bpsABCD locus was found to not be required for persistence in the upper respiratory tract of swine. Additionally, the bpsABCD locus did not affect the development of anti- Bordetella humoral immunity, did not contribute to disease severity, and did not mediate protection from complement-mediated killing. However, the bpsABCD locus was found to enhance survival in the lower respiratory tract of swine., (Copyright © 2017 American Society for Microbiology.)

Bordetella bronchiseptica produces respiratory disease primarily in mammals including humans. Although a considerably amount of research has been generated regarding lipopolysaccharide (LPS) role during infection and stimulating innate and adaptive immune response, mechanisms involved in LPS synthesis are still unknown. In this context we searched in B. bronchiseptica genome for putative glycosyltransferases. We found possible genes codifying for enzymes involved in sugar substitution of the LPS structure. We decided to analyse BB3394 to BB3400 genes, closed to a previously described LPS biosynthetic locus in B. pertussis. Particularly, conservation of BB3394 in sequenced B. bronchiseptica genomes suggests the importance of this gene for bacteria normal physiology. Deletion of BB3394 abolished resistance to naive serum as described for other LPS mutants. When purified LPS was analyzed, differences in the LPS core structure were found. Particularly, a GalNA branched sugar substitution in the core was absent in the LPS obtained from BB3394 deletion mutant. Absence of GalNA in core LPS alters immune response in vivo but is able to induce protective response against B. bronchiseptica infection., (Copyright © 2016. Published by Elsevier Ltd.)

The baboon model of Bordetella pertussis infection is the newest and most clinically accurate model of the human disease to date. However, among the 15 experimentally infected baboons in this study, a subset of baboons did not exhibit the expected high bacterial colonization levels or increase in white blood cell count. Moreover, cultures of nasopharyngeal wash samples from several baboons suggested B. bronchiseptica coinfection. Analysis of serum antibodies recognizing filamentous hemagglutinin, pertussis toxin and B. pertussis lipo-oligosaccharide indicated that several baboons had likely been previously exposed to Bordetella species and that prior exposure correlated with partial protection from B. pertussis infection. Notably, all animals with a baseline Fha titer of 5 IU/ml or below exhibited symptoms typical of the model, suggesting this value can be used as inclusion criteria for animals prior to study enrollment. While B. pertussis infection is endemic to human populations and B. bronchiseptica is common in wild small mammals, this study illustrates that baboons can readily harbor both organisms. Awareness of Bordetella species that share antigens capable of generating protective immune responses and tracking of prior exposure to those species is required for successful use of the baboon model of pertussis.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important disease around the globe. Protection against this virus remains problematic. Here, we evaluated antibody (IgG & IgA) inducibility of a heterologous PRRSV glycoprotein 5 (GP5) expressed in a live attenuated Bordetella bronchisepticaaroA mutant strain (BBS-GP5). Mice and pigs were primed with recombinant GP5 (rGP5) subcutaneously followed by boosting with live BBS-GP5. As a result, anti-GP5 IgG was induced in both mice (P<0.001) and pigs (P<0.1). Pigs were challenged with live PRRSV (VR2332). Viral RNA was found to be significantly (P<0.01) removed in the vaccinated pig group. Overall, BBS-GP5 is a good candidate as a live attenuated vaccine against PRRSV infection.

PvuII ribotyping and MLST are each highly discriminatory methods for genotyping Bordetella bronchiseptica, but a direct comparison between these approaches has not been undertaken. The goal of this study was to directly compare the discriminatory power of PvuII ribotyping and MLST, using a single set of geographically and genetically diverse strains, and to determine whether subtyping based on repeat region sequences of the pertactin gene (prn) provides additional resolution. One hundred twenty-two isolates were analyzed, representing 11 mammalian or avian hosts, sourced from the United States, Europe, Israel and Australia. Thirty-two ribotype patterns were identified; one isolate could not be typed. In comparison, all isolates were typeable by MLST and a total of 30 sequence types was identified. An analysis based on Simpson's Index of Diversity (SID) revealed that ribotyping and MLST are nearly equally discriminatory, with SIDs of 0.920 for ribotyping and 0.919 for MLST. Nonetheless, for ten ribotypes and eight MLST sequence types, the alternative method discriminates among isolates that otherwise type identically. Pairing prn repeat region typing with ribotyping yielded 54 genotypes and increased the SID to 0.954. Repeat region typing combined with MLST resulted in 47 genotypes and an SID of 0.944. Given the technical and practical advantages of MLST over ribotyping, and the nominal difference in their SIDs, we conclude MLST is the preferred primary typing tool. We recommend the combination of MLST and prn repeat region typing as a high-resolution, objective and standardized approach valuable for investigating the population structure and epidemiology of B. bronchiseptica., (Published by Elsevier B.V.)

In this study, the adjuvant effects of the extract of Cochinchina momordica seed ECMS+oil, oil alone, ECMS alone, conventional alum adjuvant on inactivated Bordetella bronchiseptica (Bb) vaccine or control using antigen alone without adjuvant were evaluated along with the underlying mechanism. The results in experiment A demonstrated that antibody levels in Bb whole cell protein in the ECMS800μg+oil group were significantly higher than in the other adjuvant groups (p<0.05) on day 21. The agglutination antibody titer was also higher than the other groups (p<0.05) on day 37. The ECMS800μg+oil group improved cellular immune responses compared to other adjuvant groups, including control using antigen alone without adjuvant and the PBS group (p<0.05). After Bb challenge, the ECMS800μg+oil group showed the highest protection rate, which was significantly higher than ECMS alone or control using antigen alone without adjuvant and the PBS group (p<0.05 and p<0.01). IgA cells in the ECMS800μg+oil group differed significantly from the IgA cells of other groups in the lungs (p<0.01). The results of cell recruitment showed that the number of lymphocytes in the ECMS400μg+oil were higher than the number of cells for other groups except the ECMS(100μg/800μg)+oil groups (p<0.05). Intermediate cells in the ECMS(100μg/400μg)+oil groups were higher than the number of cells for other groups, including the control using antigen alone group (p<0.05). Neutrophils in the ECMS(100μg/400μg/800μg)+oil groups were significantly higher than the ECMS 800μg and control using antigen alone groups (p<0.05). White blood cells in the ECMS100μg+oil group were significantly higher than the oil, ECMS800μg and control using antigen alone groups (p<0.05). IL-2 expression in the ECMS800μg+oil group was significantly higher than other groups, except for the ECMS400μg+oil group (p<0.05). IL-4 expression in the ECMS800μg+oil group was significantly higher than other groups (p<0.05). GATA3 in the ECMS800μg+oil groups was significantly higher than the oil, ECMS800μg and control using antigen alone group (p<0.05). ECMS-oil adjuvant mixture could most effectively protect B. bronchiseptica immunized rabbits and, therefore, could be an alternative way of improving B. bronchiseptica vaccination in rabbits., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In order to determine the comparative efficacy of vaccines administered intranasally or orally to protect puppies from disease subsequent to experimental infection with Bordetella bronchiseptica (Bb), a randomized controlled trial was performed using 48 approximately 8-week-old specific pathogen free, Bb naive Beagle puppies. Puppies were randomized into three groups and administered vaccines containing Bb intranasally or orally, or a placebo intranasally. Twenty-one days later, all dogs were challenge exposed via aerosol administration of Bb. Clinical signs, nasal bacterial shedding and immune responses were monitored for 28 days after challenge. Intranasally vaccinated puppies had significantly lower rates of coughing, nasal discharge, retching and sneezing (i.e. were less sick clinically) than control puppies. The distinction between the orally vaccinated puppies and the control puppies was less consistent. The orally vaccinated puppies had less coughing and less retching than the control puppies, but nasal discharge and sneezing did not differ from control animals. Orally vaccinated puppies had higher rates of coughing, nasal discharge, retching and sneezing than the intranasally vaccinated puppies. Although both intranasal and oral Bb vaccines stimulated immune responses associated with disease sparing following Bb infection, the intranasal route of delivery conferred superior clinical outcomes. The observed difference in clinical efficacy suggests the need to question the rationale for the use of currently available orally administered Bb vaccines., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Bordetella bronchiseptica can use catecholamines to obtain iron from transferrin and lactoferrin via uptake pathways involving the BfrA, BfrD, and BfrE outer membrane receptor proteins, and although Bordetella pertussis has the bfrD and bfrE genes, the role of these genes in iron uptake has not been demonstrated. In this study, the bfrD and bfrE genes of B. pertussis were shown to be functional in B. bronchiseptica, but neither B. bronchiseptica bfrD nor bfrE imparted catecholamine utilization to B. pertussis. Gene fusion analyses found that expression of B. bronchiseptica bfrA was increased during iron starvation, as is common for iron receptor genes, but that expression of the bfrD and bfrE genes of both species was decreased during iron limitation. As shown previously for B. pertussis, bfrD expression in B. bronchiseptica was also dependent on the BvgAS virulence regulatory system; however, in contrast to the case in B. pertussis, the known modulators nicotinic acid and sulfate, which silence Bvg-activated genes, did not silence expression of bfrD in B. bronchiseptica. Further studies using a B. bronchiseptica bvgAS mutant expressing the B. pertussis bvgAS genes revealed that the interspecies differences in bfrD modulation are partly due to BvgAS differences. Mouse respiratory infection experiments determined that catecholamine utilization contributes to the in vivo fitness of B. bronchiseptica and B. pertussis. Additional evidence of the in vivo importance of the B. pertussis receptors was obtained from serologic studies demonstrating pertussis patient serum reactivity with the B. pertussis BfrD and BfrE proteins., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The Type Six Secretion System (T6SS) is required for Bordetella bronchiseptica cytotoxicity, cytokine modulation, infection, and persistence. However, one-third of recently sequenced Bordetella bronchiseptica strains of the predominantly human-associated Complex IV have lost their T6SS through gene deletion or degradation. Since most human B. bronchiseptica infections occur in immunocompromised patients, we determine here whether loss of Type Six Secretion is beneficial to B. bronchiseptica during infection of immunocompromised mice. Infection of mice lacking adaptive immunity (Rag1-/- mice) with a T6SS-deficient mutant results in a hypervirulent phenotype that is characterized by high numbers of intracellular bacteria in systemic organs. In contrast, wild-type B. bronchiseptica kill their eukaryotic cellular hosts via a T6SS-dependent mechanism that prevents survival in systemic organs. High numbers of intracellular bacteria recovered from immunodeficient mice but only low numbers from wild-type mice demonstrates that B. bronchiseptica survival in an intracellular niche is limited by B and T cell responses. Understanding the nature of intracellular survival during infection, and its effects on the generation and function of the host immune response, are important to contain and control the spread of Bordetella-caused disease.

Dermonecrotic toxin (DNT) produced by Bordetella bronchiseptica (B. bronchiseptica) can cause clinical turbinate atrophy in swine and induce dermonecrotic lesions in model mice. We know that the N-terminal of DNT molecule contains the receptor-binding domain, which facilitates binding to the target cells. However, we do not know whether this domain has sufficient immunogenicity to resist B. bronchiseptica damage and thereby to develop a subunit vaccine for the swine industry. In this study, we prokaryotically expressed the recombinant N-terminal of DNT from B. bronchiseptica (named DNT-N) and prepared it for the subunit vaccine to evaluate its immunogenicity. Taishan Pinus massoniana pollen polysaccharide (TPPPS), a known immunomodulator, was used as the adjuvant to examine its immune-conditioning effects. At 49 d after inoculation, 10 mice from each group were challenged with B. bronchiseptica, and another 10 mice were intradermally challenged with native DNT, to examine the protection imparted by the vaccines. The immune parameters (T-lymphocyte counts, cytokine secretions, serum antibody titers, and survival rates) and skin lesions were determined. The results showed that pure DNT-N vaccine significantly induced immune responses and had limited ability to resist the B. bronchiseptica and DNT challenge, whereas the mice administered with TPPPS or Freund's incomplete adjuvant vaccine could induce higher levels of the above immune parameters. Remarkably, the DNT-N vaccine combined with TPPPS adjuvant protected the mice effectively to prevent B. bronchiseptica infection. Our findings indicated that DNT-N has potential for development as an effective subunit vaccine to counteract the damage of B. bronchiseptica infection, especially when used conjointly with TPPPS., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Unlabelled: Bordetella filamentous hemagglutinin (FHA), a primary component of acellular pertussis vaccines, contributes to virulence, but how it functions mechanistically is unclear. FHA is first synthesized as an ~370-kDa preproprotein called FhaB. Removal of an N-terminal signal peptide and a large C-terminal prodomain (PD) during secretion results in "mature" ~250-kDa FHA, which has been assumed to be the biologically active form of the protein. Deletion of two C-terminal subdomains of FhaB did not affect production of functional FHA, and the mutant strains were indistinguishable from wild-type bacteria for their ability to adhere to the lower respiratory tract and to suppress inflammation in the lungs of mice. However, the mutant strains, which produced altered FhaB molecules, were eliminated from the lower respiratory tract much faster than wild-type B. bronchiseptica, suggesting a defect in resistance to early immune-mediated clearance. Our results revealed, unexpectedly, that full-length FhaB plays a critical role in B. bronchiseptica persistence in the lower respiratory tract., Importance: The Bordetella filamentous hemagglutinin (FHA) is a primary component of the acellular pertussis vaccine and an important virulence factor. FHA is initially produced as a large protein that is processed during secretion to the bacterial surface. As with most processed proteins, the mature form of FHA has been assumed to be the functional form of the protein. However, our results indicate that the full-length form plays an essential role in virulence in vivo. Furthermore, we have found that FHA contains intramolecular regulators of processing and that this control of processing is integral to its virulence activities. This report highlights the advantage of studying protein maturation and function simultaneously, as a role for the full-length form of FHA was evident only from in vivo infection studies and not from in vitro studies on the production or maturation of FHA or even from in vitro virulence-associated activity assays., (Copyright © 2015 Melvin et al.)

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.

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., (Copyright © 2015 Scheller et al.)

Bordetella bronchiseptica (Bb) has long been causally associated with respiratory disease in dogs. Parenteral and intranasal vaccines for this pathogen have been in common use since their development in the late 1970s and early 1980s and recently a commercial oral Bb vaccine has become available. Overall, the literature (comprising experimental infection models and field studies) documents the efficacy of these vaccines in stimulating disease-sparing mucosal and systemic immune responses that can be associated with reduced growth of Bb in vivo. However, many of the published studies are limited by flaws in experimental design, most notably a failure to consider the biological and statistical implications of the 'pen effect'. Many questions related to the longevity of vaccine induced immunity against Bb and the impact of natural exposure on herd immunity remain unanswered., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Fucoidan is a sulfated polysaccharide derived from brown seaweed, including Fucus vesiculosus. This compound is known to have immunostimulatory effects on various types of immune cells including macrophages and dendritic cells. A recent study described the application of fucoidan as a vaccine adjuvant. Vaccination is regarded as the most efficient prophylactic method for preventing harmful or epidemic diseases. To increase vaccine efficacy, effective adjuvants are needed. In the present study, we determined whether fucoidan can function as an adjuvant using vaccine antigens. Flow cytometric analysis revealed that fucoidan increases the expression of the activation markers major histocompatibility complex class II, cluster of differentiation (CD)25, and CD69 in spleen cells. In combination with Bordetella bronchiseptica antigen, fucoidan increased the viability and tumor necrosis factor-α production of spleen cells. Furthermore, fucoidan increased the in vivo production of antigen-specific antibodies in mice inoculated with Mycoplasma hyopneumoniae antigen. Overall, this study has provided valuable information about the use of fucoidan as a vaccine adjuvant.

Bordetella bronchiseptica (Bb) whole cell bacterins have been replaced with acelluar vaccines. We evaluated the response to the acellular Bb vaccines in Bb-seropositive commingled laboratory beagles and client-owned dogs with various lifestyles and vaccination histories. A single parenteral dose of the acellular Bb vaccine resulted in consistent anamnestic IgG, and to a lesser, but notable extent, IgA, Bb-reactive antibody responses in the seropositive beagles. Associated with the increase in antibodies measured by enzyme-linked immunosorbent assay (ELISA) was an increase in the complement (C)-dependent IgG antibody mediated bactericidal effect on Bb in vitro. Antibody responses in client-owned dogs were more variable and were dependent upon the vaccination history and serological evidence of previous Bb exposure. Antibodies from vaccinated dogs recognized several Bb proteins, notably P68 (pertactin) and P220 (fimbrial hemagglutinin), the response to which has been shown to be disease-sparing in Bp infections. These antibody responses were similar to those in experimentally infected dogs and in dogs that had received a widely used whole cell bacterin.

Background: Vaccine development has largely focused on the ability of vaccines to reduce disease in individual hosts, with less attention to assessing the vaccine's effects on transmission between hosts. Current acellular vaccines against Bordetella pertussis are effective in preventing severe disease but have little effect on less severe coughing illness that can mediate transmission., Methods: Using mice that are natural host's of Bordetella bronchiseptica, we determined the effects of vaccination on shedding and transmission of this pathogen., Results: Vaccination with heat-killed whole-cell B. bronchiseptica or B. pertussis inhibited shedding of B. bronchiseptica. Differences in neutrophil and B-cell recruitment distinguished sham-vaccine from whole-cell-----vaccine responses and correlated with shedding output. Both B and T cells were essential for vaccine-induced control of shedding. Adoptive transfer of antibodies was able to limit shedding, while depletion of CD4(+) T cells led to increased shedding in vaccinated mice. Finally, whole-cell vaccination was able to prevent transmission, but an acellular vaccine that effectively controls disease failed to control shedding and transmission., Conclusions: Our results highlight discrepancies between whole-cell and acellular vaccination that could contribute to the increased incidence of B. pertussis infection since the transition to the use of acellular vaccination., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Pigs serve as a valuable animal experimental model for several respiratory pathogens, including Swine Influenza Virus (SIV) and Bordetella bronchiseptica (Bbr). To investigate the effect of SIV and Bbr coinfection on cytokine and viral RNA expression, we performed a study in which pigs were inoculated with SIV, Bbr or both pathogens (SIV/Bbr). Our results indicate that Bbr infection alters SIV clearance. Pulmonary lesions in the SIV/Bbr group were more severe when compared to SIV or Bbr groups and Bbr did not cause significant lesions. Broncho-alveolar lavage fluid (BALF) was examined for inflammatory mediators by qPCR. Interferon (IFN)-α, interleukin IL-8, IL-1 peaked in BALF at 2 DPI, while the virus titres and severity of clinical signs were maximal at the same time. Despite its increased expression in co-infected pigs, interferon-α did not enhance SIV clearance, since the viral replication was detected at the same day as the highest IFN levels. The mRNA levels for IFN-α, IL-1β and IL-8 were significantly higher in BALF of co-infected pigs and correlated with enhanced viral RNA titers in lungs, trachea and nasal swabs. Transcription of mRNA for IL-1β was stable in SIV and SIV/Bbr groups throughout all the study. In Bbr group, the levels of mRNAs for IL-1β were significantly higher at 2, 4 and 9 DPI. The mean levels of mRNAs for TNF-α were lower than the levels of other chemokines and cytokines in all infected groups. Transcript levels of IL-10 and IL-4 did not increase at each time points. Overall, SIV replication was increased by Bbr presence and the enhanced production of pro-inflammatory mediators could contribute to the exacerbated pulmonary lesions., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Bordetella bronchiseptica is pervasive in swine populations and plays multiple roles in respiratory disease. Most studies addressing virulence factors of B. bronchiseptica utilize isolates derived from hosts other than pigs in conjunction with rodent infection models. Based on previous in vivo mouse studies, we hypothesized that the B. bronchiseptica type III secretion system (T3SS) would be required for maximal disease severity and persistence in the swine lower respiratory tract. To examine the contribution of the T3SS to the pathogenesis of B. bronchiseptica in swine, we compared the abilities of a virulent swine isolate and an isogenic T3SS mutant to colonize, cause disease, and be transmitted from host to host. We found that the T3SS is required for maximal persistence throughout the lower swine respiratory tract and contributed significantly to the development of nasal lesions and pneumonia. However, the T3SS mutant and the wild-type parent are equally capable of transmission among swine by both direct and indirect routes, demonstrating that transmission can occur even with attenuated disease. Our data further suggest that the T3SS skews the adaptive immune response in swine by hindering the development of serum anti-Bordetella antibody levels and inducing an interleukin-10 (IL-10) cell-mediated response, likely contributing to the persistence of B. bronchiseptica in the respiratory tract. Overall, our results demonstrate that the Bordetella T3SS is required for maximal persistence and disease severity in pigs, but not for transmission.

Pathogen transmission cycles require many steps: initial colonization, growth and persistence, shedding, and transmission to new hosts. Alterations in the membrane components of the bacteria, including lipid A, the membrane anchor of lipopolysaccharide, could affect any of these steps via its structural role protecting bacteria from host innate immune defenses, including antimicrobial peptides and signaling through Toll-like receptor 4 (TLR4). To date, lipid A has been shown to affect only the within-host dynamics of infection, not the between-host dynamics of transmission. Here, we investigate the effects of lipid A modification in a mouse infection and transmission model. Disruption of the Bordetella bronchiseptica locus (BB4268) revealed that ArnT is required for addition of glucosamine (GlcN) to B. bronchiseptica lipid A. ArnT modification of lipid A did not change its TLR4 agonist activity in J774 cells, but deleting arnT decreased resistance to killing by cationic antimicrobial peptides, such as polymyxin B and β-defensins. In the standard infection model, mutation of arnT did not affect B. bronchiseptica colonization, growth, persistence throughout the respiratory tract, recruitment of neutrophils to the nasal cavity, or shedding of the pathogen. However, the number of bacteria necessary to colonize a host (50% infective dose [ID50]) was 5-fold higher for the arnT mutant. Furthermore, the arnT mutant was defective in transmission between hosts. These results reveal novel functions of the ArnT lipid A modification and highlight the sensitivity of low-dose infections and transmission experiments for illuminating aspects of infectious diseases between hosts. Factors such as ArnT can have important effects on the burden of disease and are potential targets for interventions that can interrupt transmission.

Transmission of pathogens has been notoriously difficult to study under laboratory conditions leaving knowledge gaps regarding how bacterial factors and host immune components affect the spread of infections between hosts. We describe the development of a mouse model of transmission of a natural pathogen, Bordetella bronchiseptica, and its use to assess the impact of host immune functions. Although B. bronchiseptica transmits poorly between wild-type mice and mice lacking other immune components, it transmits efficiently between mice deficient in Toll-Like Receptor 4 (TLR4). TLR4-mutant mice were more susceptible to initial colonization, and poorly controlled pathogen growth and shedding. Heavy neutrophil infiltration distinguished TLR4-deficient responses, and neutrophil depletion did not affect respiratory CFU load, but decreased bacterial shedding. The effect of TLR4 response on transmission may explain the extensive variation in TLR4 agonist potency observed among closely related subspecies of Bordetella. This transmission model will enable mechanistic studies of how pathogens spread from one host to another, the defining feature of infectious disease.

Background: Bordetella bronchiseptica is a common pathogenic or colonizing organism of domestic mammals. In dogs, it causes an infectious tracheobronchitis known as Kennel Cough. Human infections are unusual and almost exclusively described in immunocompromised patients who have had contact with a known animal reservoir. It is rarely reported in Cystic Fibrosis (CF), possibly hampered by low recovery from culture and organism misidentification. We describe the incidence and characteristics of B. bronchiseptica in our CF population., Methods: A retrospective cohort study was conducted of our center's CF patient population. Patients were included if they had B. bronchiseptica isolated on one or more occasion., Results: Seven children with CF isolated B. bronchiseptica on 23 occasions, frequently associated with the symptoms of a pulmonary exacerbation. Four patients required hospitalization., Conclusion: These results suggest that B. bronchiseptica may be more common than previously reported and may play a potential pathogenic role in CF., (© 2013.)

Bordetella bronchiseptica is a Gram-negative respiratory pathogen responsible for atrophic rhinitis and bronchopneumonia in swine. Several vaccines aimed at preventing B. bronchiseptica have been used, but a safe and efficient live vaccine for use in piglets remains elusive. In this study, we constructed an aroA-deleted B. bronchiseptica strain (QH0814) and evaluated its safety and protective efficiency in piglets. Lung lesion scores in QH0814-immunized piglets post-challenge were significantly lower than those in piglets immunized with the parent strain (P<0.05). Immunization with QH0814 induced a vigorous immune response, especially at the mucosal surface of the respiratory tract. IgA titers in bronchoalveolar lavage fluid (BALF) and serum were significantly higher in the QH0814-immunized group compared to the inactivated-vaccine-immunized group. Piglets immunized with QH0814 were better protected than those in the inactivated-vaccine and negative control groups. The clinical symptoms, histopathological changes and immune responses elicited in the piglets were recorded. The results of this study suggest that QH0814 was able to confer complete protection against B. bronchiseptica infection and could thus be used as a candidate attenuated live vaccine against B. bronchiseptica in piglets., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Bordetella bronchiseptica is an important pathogen causing a number of veterinary respiratory syndromes in agriculturally important and food-producing confinement-reared animals, resulting in great economic losses annually amounting to billions of euros worldwide. Currently available live vaccines are incompletely satisfactory in terms of efficacy and safety. An efficient vaccine for livestock animals would allow reducing the application of antibiotics, thereby preventing the massive release of pharmaceuticals into the environment. Here, we describe two new potential vaccine strains based on the BB7865 strain. Two independent attenuating mutations were incorporated by homologous recombination in order to make negligible the risk of recombination and subsequent reversion to the virulent phenotype. The mutations are critical for bacterial metabolism, resistance to oxidative stress, intracellular survival and in vivo persistence. The resulting double mutants BB7865 risA aroA and BB7865 risA dapE were characterized as promising vaccine candidates, which are able to confer protection against colonization of the lower respiratory tract after sublethal challenge with the wild-type strain., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

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.

Background: Studies suggest that intranasal vaccination can stimulate nonspecific immunity against agents not contained within the vaccine, but this effect is not reported for cats., Hypothesis: A modified live feline herpesvirus-1 (FHV-1) and feline calicivirus (FCV) intranasal vaccine will reduce clinical signs of disease caused by experimental infection with Bordetella bronchiseptica., Animals: Twenty specific pathogen-free 12-week-old kittens., Methods: Experimental study. Cats were randomized into 2 groups of 10 cats each. The vaccinated group was administered a single intranasal dose of a commercially available vaccine containing modified live strains of FHV-1 and FCV, and the control group remained unvaccinated. All 20 cats were administered B. bronchiseptica by nasal inoculation 7 days later and were observed daily for clinical signs of illness for 20 days., Results: In the first 10 days after B. bronchiseptica challenge, vaccinated cats were less likely to be clinically ill than control cats with a median clinical score of 0/180 (range 0-5) versus 2/180 (range 0-8) (P = .01). Nine of 10 control cats and 2 of 10 vaccinated cats were recorded as sneezing during days 1-10 after challenge (P = .006)., Conclusions and Clinical Importance: Intranasal vaccination against FHV-1 and FCV decreased signs of illness due to an infectious agent not contained in the vaccine. This nonspecific immunity could be beneficial for protection against organisms for which vaccines are not available and as protection before development of vaccine-induced humoral immunity., (Copyright © 2012 by the American College of Veterinary Internal Medicine.)

Bordetella bronchiseptica, a gram-negative bacterium, causes chronic respiratory tract infections in a wide variety of mammalian hosts, including man, and no human vaccine is currently available. Acellular pertussis vaccines protect poorly against B. bronchiseptica, although they contain cross-reactive antigens. We have recently developed Bordetella pertussis BPZE1, a novel, live attenuated pertussis vaccine, currently completing phase I clinical trials in humans, and found that it protects against both B. pertussis and Bordetella parapertussis in mice. Here, we show that a single nasal administration of BPZE1 protects mice against lethal infection with B. bronchiseptica. After challenge, the vaccinated animals displayed markedly reduced lung inflammation and tissue damage, decreased neutrophil infiltration and increased levels of CD4(+)CD25(+)FoxP3(+) regulatory T cells in the lungs compared to non-immunized mice. Depletion of these cells abolished BPZE1-induced protection, indicating that BPZE1 protects against lethal inflammation through the recruitment of regulatory T cells. In addition, the B. bronchiseptica load was significantly decreased in the vaccinated animals. Using passive transfer experiments, protection was found to be essentially cell mediated, and BPZE1-induced Th1 and Th17 T cells recognize whole B. bronchiseptica extracts, although the participation of antibodies in protection cannot be discounted. Thus, a single administration of BPZE1 can confer protection against B. bronchiseptica in mice by a dual mechanism., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Co-infections can alter the host immune responses and modify the intensity and dynamics of concurrent parasitic species. The extent of this effect depends on the properties of the system and the mechanisms of host-parasite and parasite-parasite interactions. We examined the immuno-epidemiology of a chronic co-infection to reveal the immune mediated relationships between two parasites colonising independent organs, and the within-host molecular processes influencing the dynamics of infection at the host population level. The respiratory bacterium, Bordetella bronchiseptica, and the gastrointestinal helminth, Graphidium strigosum, were studied in the European rabbit (Oryctolagus cuniculus), using long-term field data and a laboratory experiment. We found that 65% of the rabbit population was co-infected with the two parasites; prevalence and intensity of co-infection increased with rabbit age and exhibited a strong seasonal pattern with the lowest values recorded during host breeding (from April to July) and the highest in the winter months. Laboratory infections showed no significant immune-mediated effects of the helminth on bacterial intensity in the lower respiratory tract but a higher abundance was observed in the nasal cavity during the chronic phase of the infection, compared with single bacterial infections. In contrast, B. bronchiseptica enhanced helminth intensity and this was consistent throughout the 4-month trial. These patterns were associated with changes in the immune profiles between singly and co-infected individuals for both parasites. This study confirmed the general observation that co-infections alter the host immune responses but also highlighted the often ignored role of bacterial infection in helminth dynamics. Additionally, we showed that G. strigosum had contrasting effects on B. bronchiseptica colonising different parts of the respiratory tract. At the host population level our findings suggest that B. bronchiseptica facilitates G. strigosum infection, and re-infection with G. strigosum assists in maintaining bacterial infection in the upper respiratory tract and thus long-term persistence., (Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

The present authors have previously obtained the Bordetella bronchiseptica mutant BbLP39, which contains a deep-rough lipopolysaccharide (LPS) instead the wild type smooth LPS with O antigen. This mutant was found to be altered in the expression of some proteins and in its ability to colonize mouse lungs. Particularly, in BbLP39 the expression of pertactin is decreased. To differentiate the contribution of each bacterial component to the observed phenotype, here mice defective in the LPS sensing receptor TLR4 (TLR4-defective mice) were used. In contrast to wild-type mice, infection of TLR4-defective mice with BbLP39 resulted in lung infection, which persisted for more than 10 days post-challenge. Comparative analysis of the immune responses induced by purified mutant and wild type LPSs showed that the mutant LPS induced significantly higher degrees of expression of TNF-α and IL-10 mRNA than did the wild type. UV matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry analysis revealed that both LPSs had the same penta-acylated lipid A structure. However, the lipid A from BbLP39 contained pyrophosphate instead of phosphate at position 1. This structural difference, in addition to the lack of O-antigen in BbLP39, may explain the functional differences between BbLP39 and wild type strains., (© 2011 The Societies and Blackwell Publishing Asia Pty Ltd.)

Understanding seasonal changes in age-related incidence of infections can be revealing for disentangling how host heterogeneities affect transmission and how to control the spread of infections between social groups. Seasonal forcing has been well documented in human childhood diseases but the mechanisms responsible for age-related transmission in free-living and socially structured animal populations are still poorly known. Here we studied the seasonal dynamics of Bordetella bronchiseptica in a free-living rabbit population over 5 years and discuss the possible mechanisms of infection. This bacterium has been isolated in livestock and wildlife where it causes respiratory infections that rapidly spread between individuals and persist as subclinical infections. Sera were collected from rabbits sampled monthly and examined using an ELISA. Findings revealed that B. bronchiseptica circulates in the rabbit population with annual prevalence ranging between 88% and 97%. Both seroprevalence and antibody optical density index exhibited 1-year cycles, indicating that disease outbreaks were seasonal and suggesting that long-lasting antibody protection was transient. Intra-annual dynamics showed a strong seasonal signature associated with the recruitment of naive offspring during the breeding period. Infection appeared to be mainly driven by mother-to-litter contacts rather than by interactions with other members of the community. By age 2 months, 65% of the kittens were seropositive.

This prospective study evaluated seroepidemiologic features of canine respiratory coronavirus (CRCoV), canine parainfluenza virus (CPIV), and Bordetella bronchiseptica infections in dogs in an urban humane shelter and in rural/small community dog populations in western Canada. Seroprevalence of CRCoV and CPIV was low compared with other countries; seroprevalence of B. bronchiseptica was moderate to high in most populations examined. Rural dogs were 0.421 times (P ≤ 0.0001) less likely to be positive for CRCoV than dogs admitted to the shelter. There were no statistical differences in prevalence of antibodies to B. bronchiseptica and CPIV between urban and rural populations. Dogs from Fort Resolution, NWT were significantly (P < 0.05) less likely to have moderate or high antibody titers to the 3 agents than dogs in the shelter. Seroconversion to CRCoV was common in dogs in the shelter, but was not associated (P = 0.18) with respiratory disease. Antibodies to CRCoV, CPIV, or B. bronchiseptica on arrival were not significantly (P > 0.05) associated with disease-sparing after entry into the shelter.

We have provided a detailed structural analysis of porcine alveolar macrophage TLR3 extracellular domain (ECD). The porcine TLR3-ECD contains 18 leucine-rich repeats (LRRs) consisting of blocks of consensus motifs and non-consensus motifs containing insertions. Excluding the N-terminal and C-terminal LRRs, porcine TLR3 has two LRRs with insertions, resulting in one LRR of 39 amino acids and another LRR of 34 amino acids. Furthermore, we have conducted the first examination of the regulated expression of porcine alveolar macrophage TLR3 during in vivo co-infection with influenza virus and Bordetella bronchiseptica. There was a bi-phasic upregulation of porcine TLR3 during influenza virus infection (day 1 and day 10 post-infection). Co-infection resulted in an enhanced expression of porcine TLR3 only at day 1 post-infection. Interestingly, B. bronchiseptica induced an upregulation in alveolar macrophage TLR3 expression at day 10 post-infection. Based on our work and that of others, TLR3 likely plays a key role in the immune response of lung cells to influenza virus infection in several mammalian species., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Pertussis is a highly contagious respiratory disease that is especially dangerous for infants and children. Despite mass vaccination, reported pertussis cases have increased in the United States and other parts of the world, probably because of increased awareness, improved diagnostic means, and waning vaccine-induced immunity among adolescents and adults. Licensed vaccines do not kill the organism directly; the addition of a component inducing bactericidal antibodies would improve vaccine efficacy. We investigated Bordetella pertussis and Bordetella bronchiseptica LPS-derived core oligosaccharide (OS) protein conjugates for their immunogenicity in mice. B. pertussis and B. bronchiseptica core OS were bound to aminooxylated BSA via their terminal Kdo residues. The two conjugates induced similar anti-B. pertussis LPS IgG levels in mice. B. bronchiseptica was investigated because it is easier to grow than B. pertussis. Using B. bronchiseptica genetically modified strains deficient in the O-specific polysaccharide, we isolated fractions of core OS with one to five repeats of the terminal trisaccharide, having at the nonreducing end a GlcNAc or GalNAc, and bound them to BSA at different densities. The highest antibody levels in mice were elicited by conjugates containing an average of 8-17 OS chains per protein and with one repeat of the terminal trisaccharide. Conjugate-induced antisera were bactericidal against B. pertussis, and the titers correlated with ELISA-measured antibody levels (r = 0.74). Such conjugates are easy to prepare and standardize; added to a recombinant pertussis toxoid, they may induce antibacterial and antitoxin immunity.