SummaryThe attachment of pathogenic microorganisms to host cells and tissues is often mediated through the expression of surface receptors recognizing components of the extracellular matrix (ECM). Here, we investigate the ability of Borrelia spirochaetes to bind the ECM constituent, fibronectin. Borrelia lysates were separated by SDS-PAGE, transferred to nitrocellulose and probed with alkaline phosphatase-labelled fibronectin (fibronectin-AP). Five of six Borrelia species and four of eight B. burgdorferi sensu lato isolates expressed one or more fibronectin-binding proteins. Borrelia burgdorferi isolate B31 expressed a 47 kDa (P47) fibronectin-binding protein that was localized to the outer envelope based on susceptibility to proteinase K. The interaction of P47 with fibronectin was specific, and the region of fibronectin bound by P47 mapped to the gelatin/collagen binding domain. P47 was purified by affinity chromatography, digested with endoproteinase Lys-C, and the peptide fragments analysed by liquid chromatography/tandem mass spectroscopy. A search of protein databases disclosed that the P47 peptide mass profile matched that predicted for the bbk32 gene product of B. burgdorferi isolate B31. The bbk32 gene was cloned into Escherichia coli, and the ability of recombinant BBK32 to bind fibronectin and inhibit the attachment of B. burgdorferi was demonstrated. The identification of BBK32 as a receptor for fibronectin binding may enhance our understanding of the pathogenesis and chronic nature of Lyme disease.

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The identification of Brucella can be a time-consuming and labor-intensive process that places personnel at risk for laboratory-acquired infection. Here, we describe a real-time PCR assay for confirmation of presumptive Brucella isolates. The assay was designed in a multiplex format that will allow the rapid identification of Brucella spp., B. abortus, and B. melitensis in a single test.Brucellosis is a zoonotic disease that causes abortion, fetal death, and genital infections in animals. In humans, this highly diverse illness initially presents as fever, malaise, and myalgia and may later develop into a chronic illness affecting various organs and tissues. The causative agents of brucellosis are facultative intracellular gram-negative bacteria belonging to the genus Brucella. This genus has been further classified into six species according to host preference and pathogenicity. Genetic studies, however, indicate that the six classical species of Brucella represent a single genomospecies based upon DNA-DNA reassociation (14). In spite of this evidence, the conventional classification scheme has been maintained. Human brucellosis has been attributed to B. abortus, B. melitensis, B. suis, and B. canis and more recently to strains resembling Brucella isolated from marine mammals (13). Laboratory detection of Brucella and species identification is based largely on culture isolation and phenotypic characterization. This process is lengthy and labor-intensive and has been associated with a heightened risk of laboratory-acquired infections (7). To surmount these problems, nucleic acid amplification has been explored for the rapid detection and confirmation of Brucella.A number of nucleic acid sequences have been targeted for the development of Brucella genus-specific PCR assays, including 16S rRNA, the 16S-23S intergenic spacer region, omp2, and bcsp31 (1, 9, 11, 12). The most frequently described PCR target for the diagnosis of human brucellosis is the bcsp31 gene encoding a 31-kDa antigen conserved among Brucella spp. (8,9,15). PCR identification of Brucella strains at the species or biovar level has been more challenging. Recently, Redkar et al.(10) described real-time PCR assays for the detection of B. abortus, B. melitensis, and B. suis biovar 1. These PCR assays target the specific integration of IS711 elements within the genome of the respective Brucella species or biovar. The assays, however, were designed to be tested in separate PCRs. Using similar PCR targets, but in a multiplex format, we have developed a real-time triplex assay that permits rapid confirmation of Brucella spp., B. abortus, and B. melitensis isolates in a single test.The primers and TaqMan probes (Qiagen, Alameda, Calif

The identification of antigens with the capacity to induce a broad spectrum of protective immunity is an important consideration in the design of a Lyme disease vaccine. In this study, the range of protection provided by outer surface protein (Osp) A or OspC vaccination was compared. Mice actively immunized with OspA or OspC were challenged with 3 North American isolates of Borrelia burgdorferi. OspA-immunized mice were fully protected from infection with each of the isolates, whereas mice immunized with OspC were protected from infection with the homologous isolate but not with 2 heterologous isolates. Sequence analysis revealed that the ospA genes from these 3 isolates were >99% homologous, whereas the ospC genes shared only 81%-85% homology. Western blot analysis suggested antigenic heterogeneity associated with OspC but not OspA. These results indicate that genetic and antigenic heterogeneity may limit the usefulness of OspC as a vaccine constituent.