Polymorphisms in the pertussis toxin promoter in Bordetella bronchiseptica veterinary isolates from Argentina.

Five restriction patterns (including a novel one) could be defined by polymerase chain reaction-restriction fragment length polymorphism on the pertussis toxin (PT) promoter region in local veterinary isolates, suggesting that PT gene analysis is a potential molecular marker for Bordetella bronchiseptica detection and typing.

Vir90, a virulence-activated gene coding for a Bordetella pertussis iron-regulated outer membrane protein.

Bordetella pertussis undergoes phenotypic changes modulated by the bvgAS locus, which regulates the expression of many genes related to virulence and immunogenicity. We previously reported the N-terminal sequence of a 90 kDa bvg-regulated outer membrane protein (OMP) of B. pertussis (SWISS-PROT accession No. p81549), a novel potential virulence factor that we named Vir90. The open reading frames (ORFs) which potentially code for Vir90 in B. pertussis, B. parapertussis and B. bronchiseptica were identified by computer analysis of the genomic sequences available for the three Bordetella species. Nucleotide sequence analysis of the vir90 upstream region revealed the presence of a putative promoter, a BvgA binding site and a putative Fur binding site. The B. pertussis Vir90 protein showed significant homology with ferrisiderophore receptors from Gram-negative bacteria. An antiserum raised against Vir90His recombinant protein recognized the 90-kDa protein in immunoblots of OMPs from these three virulent Bordetella species. The accumulation of the Vir90 protein increased 4-fold under low iron growth conditions. Therefore, the vir90 gene is expressed in the tested species and its expression is regulated positively by the BvgAS system and negatively under high iron concentration, likely by Fur.

Characterization of intermediate phenotypes induced by chemically undefined laboratory media in virulent Bordetella bronchiseptica strains.

The expression of many virulence factors of Bordetella bronchiseptica is regulated by the bvgAS locus and reduced in response to environmental signals called modulators. Virulent strains can alternate between virulent (Bvg(+)), intermediate (Bvg(i)), and modulated (Bvg(+)mod) phenotypes. Potential vaccine antigens can be expressed by Bvg1 strains grown only in the absence of modulators. In the present study we evaluated filamentous hemagglutinin (FHA) and outer membrane protein (OMP) expression in Bvg(+) B. bronchiseptica strains grown in chemically undefined media: nutrient agar (NA), tryptic soy agar (TSA), tryptose phosphate broth (TPB), and brain-heart infusion (BHI). Our results suggest that TSA and TPB usually induce semimodulation, since Bvg(+) strains cultured in these media retained the expression of FHA and virulence-associated OMPs in the 30 kDa region, but failed to express other virulence markers such as OMPs in the regions of 90 and 200 kDa, though they expressed flagellin (avirulence marker). On the other hand, NA and BHI usually induce modulation. Thus the assayed chemically undefined media should not be used in vaccine production. Semimodulation induced by TSA and TPB can be accurately detected by SDS-PAGE Sarkosyl-insoluble OMP-enriched profiles. The reduction or absence of OMPs in the regions of 90 and 200 kDa is the most sensitive marker, and in some cases the presence of flagellin in intermediate profiles is another trait of the Bvg(i) phenotypes. Therefore these markers could be useful for selecting media for vaccine production. We also characterized the phenotype of Bvg(+) strains grown in Stainer-Scholte broth, an expensive medium, with and without glutathione, and we have detected no differences; this is the first attempt to reduce the cost of a Bordetella growth medium for veterinary vaccine production.