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The molecular and antigenic variabilities of BmpA (P39) among European isolates of Borrelia burgdorferi were analyzed. The bmpA sequences of 12 isolates representing all three species of B. burgdorferi sensu lato pathogenic for humans were amplified by PCR, cloned, and sequenced. The BmpA protein of Borrelia garinii is heterogeneous, with an amino acid sequence identity ranging from 91 to 97%, whereas the BmpA proteins of Borrelia afzelii and B. burgdorferi sensu stricto strains appear to be highly conserved (>98.5% intraspecies identity). The interspecies identities ranged from 86 to 92%. Cluster analysis of BmpA reflected the subdivision of B. burgdorferi sensu lato isolates into the three species as well as a considerable heterogeneity among B. garinii strains. The BmpA protein of each species of B. burgdorferi sensu lato was recombinantly expressed in Escherichia coli, purified, and used to generate monoclonal antibodies. Seven BmpA-specific antibodies were identified; six of them recognized conserved epitopes of all three species, whereas one was specific for BmpA of B. afzelii and B. garinii. A monoclonal antibody (H1141) recommended by the Centers for Disease Control and Prevention for use in the standardization of immunoblots showed strong reactivity with BmpA of B. burgdorferi sensu stricto but no or only weak reactivity with BmpA of B. garinii and B. afzelii, respectively. Sera from 86 European patients with Lyme borreliosis in different stages and 73 controls were tested in immunoglobulin G (IgG) and IgM immunoblots with the recombinant BmpA proteins of the three species, revealing specificities of 98.6 to 100%. IgM antibodies against recombinant BmpA were only rarely detected (1.1 to 8.1%). With the BmpA proteins of B. afzelii and B. garinii, sensitivities for the IgG test (sera from stages I to III) were 36.0 and 34.9%, respectively, in contrast to 13.9% with BmpA of B. burgdorferi sensu stricto. Therefore, we recommend that recombinant BmpA of B. afzelii or B. garinii should be used solely, or in addition to B. burgdorferi sensu stricto BmpA, in serodiagnostic tests for Lyme borreliosis in Europe.