Project description:B. parapertussis is a whooping cough etiological agent with the ability to evade the immune response induced by pertussis vaccines. We previously demonstrated that in the absence of opsonic antibodies B. parapertussis hampers phagocytosis by neutrophils and macrophages and, when phagocytosed, blocks intracellular killing by interfering with phagolysosomal fusion. But neutrophils can kill and/or immobilize extracellular bacteria through non-phagocytic mechanisms such as degranulation and neutrophil extracellular traps (NETs). In this study we demonstrated that B. parapertussis also has the ability to circumvent these two neutrophil extracellular bactericidal activities. The lack of neutrophil degranulation was found dependent on the O antigen that targets the bacteria to cell lipid rafts, eventually avoiding the fusion of nascent phagosomes with specific and azurophilic granules. IgG opsonization overcame this inhibition of neutrophil degranulation. We further observed that B. parapertussis did not induce NETs release in resting neutrophils and inhibited NETs formation in response to phorbol myristate acetate (PMA) stimulation by a mechanism dependent on adenylate cyclase toxin (CyaA)-mediated inhibition of reactive oxygen species (ROS) generation. Thus, B. parapertussis modulates neutrophil bactericidal activity through two different mechanisms, one related to the lack of proper NETs-inducer stimuli and the other one related to an active inhibitory mechanism. Together with previous results these data suggest that B. parapertussis has the ability to subvert the main neutrophil bactericidal functions, inhibiting efficient clearance in non-immune hosts.
Project description:Pertactin is an outer membrane protein expressed by Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica that induces protective immunity to Bordetella infections. The immunodominant and immunoprotective epitopes of pertactin include two repeated regions, I and II. Comparison of these two repeated regions showed that B. parapertussis pertactin is invariant, whereas B. pertussis pertactin varies mostly in region I and B. bronchiseptica pertactin varies in both repeated regions I and II, but mostly in region II. These differences may result from specific characteristics of these Bordetella species.
Project description:Background and objectiveRapid diagnosis of pertussis is important for the timely isolation of the infection source and early prevention measures among the contact persons, especially among non-vaccinated infants for whom pertussis is life-threatening.Materials and methodsTargets IS481, IS1001, BP0026 and human GAPDH gene were used to develop a multiplex real-time PCR assay based on the TaqMan technology for detection and identification of Bordetella pertussis and Bordetella parapertussis in clinical samples. A total of 121 human clinical specimens obtained within 2012-2013 were used to evaluate the multiplex real-time PCR assay. Clinical specimens were also tested for culture and conventional PCR. Sensitivity and specificity for culture, conventional PCR, and multiplex real-time PCR were measured in comparison with a clinical standard for B. pertussis infection.ResultsThe lower limit of detection (LLOD) of the multiplex assay was similar to the LLOD of each target in an individual assay format, which was approximately 1 genomic equivalent per reaction for IS481, IS1001 and 10 genomic equivalents per reaction for BP0026 target. When the B. pertussis assays were compared with a clinical standard for B. pertussis infection, sensitivity was 5, 59 and 89% the specificity was 100, 100 and 100% for culture, conventional PCR, and multiplex real-time PCR, respectively.ConclusionsDeveloped multiplex real-time PCR offers a fast tool with high sensitivity and specificity for the diagnosis of B. pertussis and B. parapertussis infections which is suitable for implementation in a routine laboratory diagnostics.
Project description:Whooping cough's primary etiological agent is Bordetella pertussis. The closely related Bordetella parapertussis rarely causes severe disease. Here we report an unusual case of bacteremia caused by B. parapertussis, review the literature, and characterize the genomic sequence of the bacterial isolate in comparison with B. parapertussis isolates from respiratory infections.
Project description:By analysis of repetitive DNA in Bordetella parapertussis, an insertion sequence element, designated IS1001, was identified. Sequence analysis revealed that IS1001 comprised 1,306 bp and contained inverted repeats at its termini. Furthermore, several open reading frames that may code for transposition functions were identified. The largest open reading frame coded for a protein comprising 406 amino acid residues and showed homology to TnpA, which is encoded by an insertion sequence element (IS1096) found in Mycobacterium smegmatis. Examination of flanking sequences revealed that insertion of IS1001 occurs preferentially in stretches of T's or A's and results in a duplication of target sequences of 6 to 8 bases. IS1001 was found in about 20 copies in 10 B. parapertussis strains analyzed. No restriction fragment length polymorphism was observed in B. parapertussis when IS1001 was used as a probe. An insertion sequence element similar or identical to IS1001 was found in B. bronchiseptica strains isolated from pigs and a rabbit. In these strains, about five copies of the IS1001-like element were present at different positions in the bacterial chromosome. Neither B. pertussis nor B. bronchiseptica strains isolated from humans and dogs contained an IS1001-like element. Therefore, IS1001 may be used as a specific probe for the detection of B. parapertussis in human clinical samples.