Bordetella avium virulence measured in vivo and in vitro.
ABSTRACT: Bordetella avium causes an upper-respiratory-tract disease called bordetellosis in birds. Bordetellosis shares many of the clinical and histopathological features of disease caused in mammals by Bordetella pertussis and Bordetella bronchiseptica. In this study we determined several parameters of infection in the domestic turkey, Meleagris galapavo, and compared these in vivo findings with an in vitro measure of adherence using turkey tracheal rings. In the in vivo experiments, we determined the effects of age, group size, infection duration, and interindividual spread of B. avium. Also, the effect of host genetic background on susceptibility was tested in the five major commercial turkey lines by infecting each with the parental B. avium strain and three B. avium insertion mutants. The mutant strains lacked either motility, the ability to agglutinate guinea pig erythrocytes, or the ability to produce dermonecrotic toxin. The susceptibilities of 1-day-old and 1-week-old turkeys to B. avium were the same, and challenge group size (5, 8, or 10 birds) had no effect upon the 50% infectious dose. Two weeks between inoculation and tracheal culture was optimal, since an avirulent mutant (unable to produce dermonecrotic toxin) persisted for a shorter time. Communicability of the B. avium parental strain between confined birds was modest, but a nonmotile mutant was less able to spread between birds. There were no host-associated differences in susceptibility to the parental strain and the three B. avium mutant strains just mentioned: in all turkey lines tested, the dermonecrotic toxin- and hemagglutination-negative mutants were avirulent whereas the nonmotile mutants showed no loss of virulence. Interestingly, the ability of a strain to cause disease in vivo correlated completely with its ability to adhere to ciliated tracheal cells in vitro.
Project description:Bordetella avium causes bordetellosis in birds, a disease similar to whooping cough caused by Bordetella pertussis in children. B. avium agglutinates guinea pig erythrocytes via an unknown mechanism. Loss of hemagglutination ability results in attenuation. We report the use of transposon mutagenesis to identify two genes required for hemagglutination. The genes (hagA and hagB) were adjacent and divergently oriented and had no orthologs in the genomes of other Bordetella species. Construction of in-frame, unmarked mutations in each gene allowed examination of the role of each in conferring erythrocyte agglutination, explanted tracheal cell adherence, and turkey poult tracheal colonization. In all of the in vitro and in vivo assays, the requirement for the trans-acting products of hagA and hagB (HagA and HagB) was readily shown. Western blotting, using antibodies to purified HagA and HagB, revealed proteins of the predicted sizes of HagA and HagB in an outer membrane-enriched fraction. Antiserum to HagB, but not HagA, blocked B. avium erythrocyte agglutination and explanted turkey tracheal ring binding. Bioinformatic analysis indicated the similarity of HagA and HagB to several two-component secretory apparatuses in which one product facilitates the exposition of the other. HagB has the potential to serve as a useful immunogen to protect turkeys against colonization and subsequent disease.
Project description:Bordetellosis is an upper respiratory disease of turkeys caused by Bordetella avium in which the bacteria attach specifically to ciliated respiratory epithelial cells. Little is known about the mechanisms of pathogenesis of this disease, which has a negative impact in the commercial turkey industry. In this study, we produced a novel explant organ culture system that was able to successfully reproduce pathogenesis of B. avium in vitro, using tracheal tissue derived from 26 day-old turkey embryos. Treatment of the explants with whole cells of B. avium virulent strain 197N and culture supernatant, but not lipopolysaccharide (LPS) or tracheal cytotoxin (TCT), specifically induced apoptosis in ciliated cells, as shown by annexin V and TUNEL staining. LPS and TCT are known virulence factors of Bordetella pertussis, the causative agent of whooping cough. Treatment with whole cells of B. avium and LPS specifically induced NO response in ciliated cells, shown by uNOS staining and diaphorase activity. The explant system is being used as a model to elucidate specific molecules responsible for the symptoms of bordetellosis.
Project description:Bordetellosis or turkey coryza, caused by Bordetella avium, has been an issue for turkey industry since its first description in 1967 when it was reported for the first time. Bordetella avium causes a highly contagious upper respiratory disease in turkeys. Therefore, this study aimed to isolate and characterize this species from commercial and backyard turkeys in Tehran, Isfahan, and Northern provinces of Iran. For the purpose of the study, 625 tracheal swabs were taken from 425 commercial poults and 200 backyard poults aged 2-6 weeks from September 2016 to September 2018. The swabs were immediately plated on MacConkey and blood agar plates and then pooled (5 swabs/pool) in tubes, containing 2 mL distilled water, to perform direct polymerase chain reaction (PCR) for the identification of B. avium. A total of 17 swab pools were found to be positive for B. avium. A subset of seven positive samples were sequenced for the flanking region of piuA gene. The analysis of the sequences indicated that the sequences were 98%, 96%, and 98% similar to B. avium 197N (AM167904.1), 4142 (AY925058.1), and 4156 (AY925068.1) sequences, respectively. To the best of our knowledge, the current study is the first attempt toward the molecular detection and characterization of B. avium in Iran. It is highly recommended to perform further studies to isolate, characterize, and differentiate the regional isolates in order to help the developing turkey industry of Iran meet the increasing demands for protein in the diet of the citizenry.
Project description:Bordetella avium causes an upper respiratory tract disease (bordetellosis) in avian species. Commercially raised turkeys are particularly susceptible. Like other pathogenic members of the genus Bordetella (B. pertussis and B. bronchiseptica) that infect mammals, B. avium binds preferentially to ciliated tracheal epithelial cells and produces similar signs of disease. These similarities prompted us to study bordetellosis in turkeys as a possible nonmammalian model for whooping cough, the exclusively human childhood disease caused by B. pertussis. One impediment to accepting such a host-pathogen model as relevant to the human situation is evidence suggesting that B. avium does not express a number of the factors known to be associated with virulence in the other two Bordetella species. Nevertheless, with signature-tagged mutagenesis, four avirulent mutants that had lesions in genes orthologous to those associated with virulence in B. pertussis and B. bronchiseptica (bvgS, fhaB, fhaC, and fimC) were identified. None of the four B. avium genes had been previously identified as encoding factors associated with virulence, and three of the insertions (in fhaB, bvgS, and fimC) were in genes or gene clusters inferred as being absent or incomplete in B. avium, based upon the lack of DNA sequence similarities in hybridization studies and/or the lack of immunological cross-reactivity of the putative products. We further found that the genotypic arrangements of most of the B. avium orthologues were very similar in all three Bordetella species. In vitro tests, including hemagglutination, tracheal ring binding, and serum sensitivity, helped further define the phenotypes conferred by the mutations. Our findings strengthen the connection between the causative agents and the pathogenesis of bordetellosis in all hosts and may help explain the striking similarities of the histopathologic characteristics of this upper airway disease in avian and mammalian species.
Project description:Bordetella avium continues to be an economic issue in the turkey industry as the causative agent of bordetellosis, which often leads to serious secondary infections. This study presents a broad characterization of the antibiotic resistance patterns in this diverse collection of B. avium strains collected over the past thirty years. In addition, the plasmid basis for the antibiotic resistance was characterized. The antibiotic resistance pattern allowed the development of a novel enrichment culture method that was subsequently employed to gather new isolates from diseased turkeys and a healthy sawhet owl. While a healthy turkey flock was shown to seroconvert by four weeks-of-age, attempts to culture B. avium from healthy turkey poults were unsuccessful. Western blot of B. avium strains using pooled serum from diseased and healthy commercial turkey flocks revealed both antigenic similarities and differences between strains. In sum, the work documents the continued exposure of commercial turkey flocks to B. avium and the need for development of an effective, inexpensive vaccine to control spread of the disease.
Project description:We isolated two insertion mutants of Bordetella avium that exhibited a peculiar clumped-growth phenotype and found them to be attenuated in turkey tracheal colonization. The mutants contained transposon insertions in homologues of the wlbA and wlbL genes of Bordetella pertussis. The wlb genetic locus of B. pertussis has been previously described as containing 12 genes involved in lipopolysaccharide (LPS) biosynthesis. Polyacrylamide gel analysis of LPS from B. avium wlbA and wlbL insertion mutants confirmed an alteration in the LPS profile. Subsequent cloning and complementation of the wlbA and wlbL mutants in trans with a recombinant plasmid containing the homologous wlb locus from B. avium eliminated the clumped-growth phenotype and restored the LPS profile to that of wild-type B. avium. Also, a parental level of tracheal colonization was restored to both mutants by the recombinant plasmid. Interestingly, complementation of the wlbA and wlbL mutants with a recombinant plasmid containing the heterologous wlb locus from B. pertussis, B. bronchiseptica, or Bordetella parapertussis eliminated the clumped-growth phenotype and resulted in a change in the LPS profile, although not to that of wild-type B. avium. The mutants also acquired resistance to a newly identified B. avium-specific bacteriophage, Ba1. Complementation of both wlbA and wlbL mutants with the homologous wlb locus of B. avium, but not the heterologous B. pertussis locus, restored sensitivity to Ba1. Complementation of the wlbL mutant, but not the wlbA mutant, with the heterologous wlb locus of Bordetella bronchiseptica or B. parapertussis restored partial sensitivity to Ba1. Comparisons of the LPS profile and phage sensitivity of the mutants upon complementation by wlb loci from the heterologous species and by B. avium suggested that phage sensitivity required the presence of O-antigen. At the mechanistic level, both mutants showed a dramatic decrease in serum resistance and a decrease in binding to turkey tracheal rings in vitro. In the case of serum resistance, complementation of both mutants with the homologous wlb locus of B. avium restored serum resistance to wild-type levels. However, in the case of epithelial cell binding, only complementation of the wlbA mutant completely restored binding to wild-type levels (binding was only partially restored in the wlbL mutant). This is the first characterization of LPS mutants of B. avium at the genetic level and the first report of virulence changes by both in vivo and in vitro measurements.
Project description:Bordetella avium is the etiologic agent of turkey coryza or bordetellosis, a respiratory disease responsible for substantial economic losses to the turkey industry. At present, identification of this bacterium relies on isolation and biochemical testing. Although a PCR for the detection of B. avium was proposed a number of years ago, lack of analytical verification precludes its use as a diagnostic tool. Furthermore, a number of details pertaining to the reaction conditions used are missing or unclear. In the present study we have identified an optimal set of PCR conditions for use with the previously described primer pair and determined the limit of detection under these conditions to be approximately 20 pg. Assay sensitivity is 100%, based on an analysis of 72 B. avium isolates from diverse geographic locations and covering a time span of at least 25 years. Evaluation of a separate group of 87 bacterial isolates from poultry, comprising both gram-positive and gram-negative commensals and pathogens representing 11 genera, demonstrated an assay specificity of 98.8%. Reproducibility is 100% using either purified genomic DNA or boiled cell lysates less than 3 days old. Sequence analysis of the B. avium PCR amplicons identified only three occasional sequence polymorphisms. These data indicate the B. avium PCR assay can provide clinically significant results.
Project description:All members of the genus Bordetella and Pasteurella multocida (a gram-negative bacillus genetically unrelated to Bordetella spp., yet often sharing the same ecological niche) produce a dermonecrotic toxin (DNT). The amount of toxin produced and the time required for appearance of the lesions are identical for Bordetella pertussis, B. parapertussis, and B. bronchiseptica but different for P. multocida and B. avium. DNT has been reported to act by promoting vasoconstriction; however, vasoactive compounds (verapamil, prazosin, hydralazine, tolazoline, or isoxsuprine) are able to reverse the action of the toxin only slightly. Vasoconstrictors (atropine, serotonin, epinephrine, or endothelin) did not produce DNT-like lesions. We have characterized a region of DNA essential for DNT expression. We have determined by Southern analysis that the restriction map of the DNT gene is nearly identical in B. pertussis, B. parapertussis, and B. bronchiseptica, but the sequences are not present in toxigenic B. avium and P. multocida strains. A gentamicin resistance-origin of transfer cassette cloned into a 1.8-kb NotI-BamHI fragment results in constructs which can be mobilized and recombined into the Bordetella chromosome, rendering the resultant B. pertussis, B. parapertussis, and B. bronchiseptica strains negative for DNT. A 5-kb BamHI-ApaI fragment from the B. pertussis chromosome was sequenced and revealed homology to the Escherichia coli CNF1 (cytotoxic necrotizing factor 1) toxin.
Project description:Bordetella avium is the causative agent of bordetellosis, which remains to be the cause of severe losses in the turkey industry. Given the lack of vaccines that can provide good protection, developing a novel vaccine against B. avium infection is crucial. In this study, we constructed a eukaryotic expression plasmid, which expressed the outer membrane protein A (ompA) of B. avium, to prepare a B. avium recombinant ompA-DNA vaccine. Three concentrations (low, middle, and high) of Taishan Pinus massoniana pollen polysaccharides (TPPPS), a known immunomodulator, were used as adjuvants, and their immune conditioning effects on the developed DNA vaccine were examined. The pure ompA-DNA vaccine, Freund's incomplete adjuvant ompA-DNA vaccine, and the empty plasmid served as the controls. The chickens in each group were separately inoculated with these vaccines three times at 1, 7, and 14 days old. Dynamic changes in antibody production, cytokine secretion, and lymphocyte count were then determined from 7 to 49 days after the first inoculation. Protective rates of the vaccines were also determined after the third inoculation. Results showed that the pure DNA vaccine obviously induced the production of antibodies, the secretion of cytokines, and the increase in CD(4+) and CD(8+) T lymphocyte counts in peripheral blood, as well as provided a protective rate of 50% to the B. avium-challenged chickens. The chickens inoculated with the TPPPS adjuvant ompA-DNA vaccine and Freund's adjuvant ompA-DNA vaccine demonstrated higher levels of immune responses than those inoculated with pure ompA-DNA vaccine, whereas only the ompA-DNA vaccine with 200 mg/mL TPPPS completely protected the chickens against B. avium infection. These findings indicate that the B. avium ompA-DNA vaccine combined with TPPPS is a potentially effective B. avium vaccine.
Project description:Three gene libraries of Bordetella avium 197 DNA were prepared in Escherichia coli LE392 by using the cosmid vectors pCP13 and pYA2329, a derivative of pCP13 specifying spectinomycin resistance. The cosmid libraries were screened with convalescent-phase anti-B. avium turkey sera and polyclonal rabbit antisera against B. avium 197 outer membrane proteins. One E. coli recombinant clone produced a 56-kDa protein which reacted with convalescent-phase serum from a turkey infected with B. avium 197. In addition, five E. coli recombinant clones were identified which produced B. avium outer membrane proteins with molecular masses of 21, 38, 40, 43, and 48 kDa. At least one of these E. coli clones, which encoded the 21-kDa protein, reacted with both convalescent-phase turkey sera and antibody against B. avium 197 outer membrane proteins. The gene for the 21-kDa outer membrane protein was localized by Tn5seq1 mutagenesis, and the nucleotide sequence was determined by dideoxy sequencing. DNA sequence analysis of the 21-kDa protein revealed an open reading frame of 582 bases that resulted in a predicted protein of 194 amino acids. Comparison of the predicted amino acid sequence of the gene encoding the 21-kDa outer membrane protein with protein sequences in the National Biomedical Research Foundation protein sequence data base indicated significant homology to the OmpA proteins of Shigella dysenteriae, Enterobacter aerogenes, E. coli, and Salmonella typhimurium and to Neisseria gonorrhoeae outer membrane protein III, Haemophilus influenzae protein P6, and Pseudomonas aeruginosa porin protein F. The gene (ompA) encoding the B. avium 21-kDa protein hybridized with 4.1-kb DNA fragments from EcoRI-digested, chromosomal DNA of Bordetella pertussis and Bordetella bronchiseptica and with 6.0- and 3.2-kb DNA fragments from EcoRI-digested, chromosomal DNA of B. avium and B. avium-like DNA, respectively. A 6.75-kb DNA fragment encoding the B. avium 21-kDa protein was subcloned into the Asd+ vector pYA292, and the construct was introduced into the avirulent delta cya delta crp delta asd S. typhimurium chi 3987 for oral immunization of birds. The gene encoding the 21-kDa protein was expressed equivalently in B. avium 197, delta asd E. coli chi 6097, and S. typhimurium chi 3987 and was localized primarily in the cytoplasmic membrane and outer membrane. In preliminary studies on oral inoculation of turkey poults with S. typhimurium chi 3987 expressing the gene encoding the B. avium 21-kDa protein, it was determined that a single dose of the recombinant Salmonella vaccine failed to elicit serum antibodies against the 21-kDa protein and challenge with wild-type B. avium 197 resulted in colonization of the trachea and thymus with B. avium 197.