Optimized ribotyping protocol applied to Hungarian Bordetella bronchiseptica isolates: identification of two novel ribotypes
ABSTRACT We reported previously that ribotype patterns generated with PvuII and a probe derived from the Escherichia coli rrnB gene could be used to differentiate isolates of Bordetella bronchiseptica. In the present study we report modifications made to the original ribotyping procedure that permit detection in the formerly characterized isolates of an additional 8 fragments with homology to rrnB. Ribotypes were redefined to include these fragments. Although this modification did not permit the detection of novel ribotypes from the previously characterized isolates, it did result in a more accurate reclassification of five of these isolates to other existing ribotypes. It was hypothesized that the additional fragments could form the basis for novel ribotypes in future analyses, and this was supported by the subsequent evaluation of 101 previously uncharacterized pig, rabbit, and dog B. bronchiseptica isolates from Hungary. A total of six different patterns were detected from this group, including two previously not identified that were designated ribotypes 17 and 18. The profile of ribotype 17 includes a novel fragment not associated with any other ribotype. A subset of the fragments constituting ribotype 18, essential for its differentiation from other ribotypes, is only detectable under the modified conditions reported here. Hungarian swine isolates are highly clonal, since 98.2% were identified as ribotype 3. Similarly, 83.7% of rabbit isolates from Hungary are also ribotype 3. Cluster analysis revealed that despite the existence of numerous ribotypes, B. bronchiseptica isolates display limited heterogeneity. The ability to detect additional ribotypes under the modified conditions described in this study strengthens the usefulness of ribotyping as an epidemiologic tool.
Article: Comparative sequence analysis of Bordetella bronchiseptica pertactin gene (prn) repeat region variants in swine vaccines and field isolates.[show abstract] [hide abstract]
ABSTRACT: The product of the Bordetella bronchiseptica pertactin gene, prn, has been implicated as an adhesin and a protective immunogen in swine. Recent studies demonstrate prn sequence heterogeneity in swine isolates and vaccine strains within and surrounding the region 1 amino acid repeat GGXXP(n) and the region 2 amino acid repeat PQP(n). However, only a few isolates have been evaluated. Allelic variation between vaccine strains and field isolates may affect vaccine efficacy, since region 2 is known to encode an immunodominant protective epitope. In the present study, the DNA and predicted amino acid sequences of the pertactin repeat regions from a collection of 81 recent swine field isolates and 5 vaccine strains from the United States were determined. Two region 1 variants and four region 2 variants, one of which has not been previously reported, were identified, comprising four pertactin types. Four vaccines are derived from strains with a region 1 variant identical to that found in the majority of field isolates. However, only two vaccines possess the most commonly identified sequence in region 2, while two others contain a variant found in only one other swine isolate. Ribotype analysis demonstrated that although vaccines containing the novel region 2 variant fall within the same major cluster as other common swine ribotypes, they are less closely related. No relationship was observed between pertactin type and ribotype.Vaccine 12/2004; 23(1):48-57. · 3.77 Impact Factor
Article: Molecular and antigenic characterization of Bordetella bronchiseptica isolated from a wild southern sea otter (Enhydra lutris nereis) with severe suppurative bronchopneumonia.[show abstract] [hide abstract]
ABSTRACT: Bordetella bronchiseptica was isolated in pure culture from the lung, abdomen, and intestine of a wild free-ranging southern sea otter (Enhydra lutris nereis) with severe, suppurative bronchopneumonia. Immunohistochemistry, using antiserum raised to B. bronchiseptica, revealed strong positive staining of bacteria attached to bronchial ciliated epithelia as well as scattered positive staining in affected alveoli. Western blot analysis demonstrated that virulence factors, filamentous hemagglutinin, pertactin, and adenylate cyclase toxin are produced by the sea otter B. bronchiseptica isolate. Ribotype analysis using Pvu II restriction digests indicated that this isolate is most similar to strains commonly obtained in domestic dogs and cats.Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 12/2003; 15(6):570-4. · 1.21 Impact Factor
Article: Contribution of Bordetella bronchiseptica filamentous hemagglutinin and pertactin to respiratory disease in swine.[show abstract] [hide abstract]
ABSTRACT: Bordetella bronchiseptica is pervasive in swine populations and plays multiple roles in respiratory disease. Most studies addressing virulence factors of B. bronchiseptica are based on isolates derived from hosts other than pigs. Two well-studied virulence factors implicated in the adhesion process are filamentous hemagglutinin (FHA) and pertactin (PRN). We hypothesized that both FHA and PRN would serve critical roles in the adhesion process and be necessary for colonization of the swine respiratory tract. To investigate the role of FHA and PRN in Bordetella pathogenesis in swine, we constructed mutants containing an in-frame deletion of the FHA or the PRN structural gene in a virulent B. bronchiseptica swine isolate. Both mutants were compared to the wild-type swine isolate for their ability to colonize and cause disease in swine. Colonization of the FHA mutant was lower than that of the wild type at all respiratory tract sites and time points examined and caused limited to no disease. In contrast, the PRN mutant caused similar disease severity relative to the wild type; however, colonization of the PRN mutant was reduced relative to the wild type during early and late infection and induced higher anti-Bordetella antibody titers. Together, our results indicate that despite inducing different pathologies and antibody responses, both FHA and PRN are necessary for optimal colonization of the swine respiratory tract.Infection and immunity 03/2009; 77(5):2136-46. · 4.21 Impact Factor