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A genome walking technique was applied to borrelial DNA to clone the gene encoding decorin-binding protein B (DbpB) in Borrelia garinii and B. afzelii. Sequence analysis showed 62–67% identity of the predicted amino acid sequences of DbpB between the B. afzelii and B. garinii strains and B. burgdorferi sensu stricto. Within subspecies, the sequences were 99–100% identical. The respective recombinant DbpBs (rDbpBs) were produced and tested as antigens in an enzyme-linked immunosorbent assay (ELISA) for Lyme borreliosis (LB). In IgG ELISA, with rDbpBs as antigens, 11 (73%) of 15 adult patients with Lyme arthritis and 9 (64%) of 14 with neuroborreliosis were positive. Of children with Lyme arthritis, 40 (77%) of 52 were positive. All adult and paediatric patients with disseminated LB had high titres of anti-flagellin IgG antibodies. Seropositivity against rDbpB from B. garinii predominated, 39 (65%) of 60 of the positive samples reacting with rDbpB from B. garinii. In patients with erythema migrans, IgM antibodies to rDbpB were detected in 1 (4%) of 23 and IgG antibodies in 6 (26%) of 23. These results indicate that DbpB may be a useful antigen in the IgG serology for disseminated LB. The high inter-species sequence heterogeneity observed indicates that a combination of the variant DbpBs should be included in the antigen set to cover all the relevant borrelial subspecies in the serodiagnosis of LB.