ABSTRACT We have studied the immune response to a variable surface-exposed loop region of the P66 outer membrane protein from Borrelia burgdorferi sensu lato by using an enzyme immunoassay. Lyme borreliosis populations found in North America and Sweden were preferentially more seroreactive to P66 from their respective regional species, namely, B. burgdorferi sensu stricto and B. garinii and B. afzelii, respectively.

To elucidate antigens present on the bacterial surface of Borrelia burgdorferi sensu lato that may be involved in pathogenesis, we characterized a protein, P13, with an apparent molecular mass of 13 kDa. The protein was immunogenic and was expressed in large amounts during in vitro cultivation compared to other known antigens. An immunofluorescence assay, immunoelectron microscopy, and protease sensitivity assays indicated that P13 is surface exposed. The deduced sequence of the P13 peptide revealed a possible signal peptidase type I cleavage site, and computer analysis predicted that P13 is an integral membrane protein with three transmembrane-spanning domains. Mass spectrometry, in vitro translation, and N- and C-terminal amino acid sequencing analyses indicated that P13 was posttranslationally processed at both ends and modified by an unknown mechanism. Furthermore, p13 belongs to a gene family with five additional members in B. burgdorferi sensu stricto. The p13 gene is located on the linear chromosome of the bacterium, in contrast to five paralogous genes, which are located on extrachromosomal plasmids. The size of the p13 transcript was consistent with a monocistronic transcript. This new gene family may be involved in functions that are specific for this spirochete and its pathogenesis.

ABSTRACTThis is the first report on the isolation of Lyme diseaseBorreliafrom seabirds on the Faeroe Islands and the characteristics of its enzootic cycle. The major components of theBorreliacycle include the puffin (Fratercula arctica) as the reservoir andIxodes uriaeas the vector. The importance of this cycle and its impact on the spread of human Lyme borreliosis have not yet been established.Borreliaspirochetes isolated from 2 of 102 sampled puffins were compared to the borreliae previously obtained from seabird ticks,I. uriae. Therrf-rrlintergenic spacer and therrsand theospCgenes were sequenced and a series of phylogenetic trees were constructed. Sequence data and restriction fragment length polymorphism analysis grouped the strains together withBorrelia garinii. In a seroepidemiological survey performed with residents involved in puffin hunting on the Faeroe Islands, 3 of 81 serum samples were found to be positive by two commonly used clinical tests: a flagellin-based enzyme-linked immunosorbent assay (ELISA) and Western blotting. These three positive serum samples also had high optical density values in a whole-cell ELISA. The finding of seropositive Faeroe Islanders who are regularly exposed toI. uriaeindicate that there may be a transfer ofB. gariniiby this tick species to humans.

A chromosomally encoded 66-kDa protein (P66) of Borrelia spp. that cause Lyme disease has previously been shown to be associated with the spirochetal outer membrane. A topological model of P66 predicts a surface-exposed fragment which links the N- and C-terminal intramembranous domains of the protein (J. Bunikis, L. Noppa, and S. Bergström, FEMS Microbiol. Lett. 131:139-145, 1995). In the present study, an immunogenic determinant of P66 was identified by a comparison of the immunoreactivities of different fragments of P66 generated either by proteolytic treatment of intact spirochetes or as recombinant proteins expressed in Escherichia coli. The immune response to P66 during natural infection was found to be directed against the predicted surface domain which comprises amino acids at positions 454 through 491. A sequence comparison revealed considerable polymorphism of the surface domains of P66 proteins of different Lyme disease-causing Borrelia species. Five sequence patterns of this domain were observed in the B. garinii strains studied. In contrast, sequences of the relevant part of P66 of the B. afzelii and B. burgdorferi sensu stricto isolates studied were identical within the respective species. In immunoblotting, 5 of 17 (29.4%) sera from North American patients with early disseminated or persistent Lyme disease reacted against P66 of B. burgdorferi sensu stricto B31. These sera, however, failed to recognize P66 of B. afzelii and B. garinii, as well as an analog of P66 in the relapsing fever agent, B. hermsii. In conclusion, the topological model of P66 is supported by the demonstration of an apparent surface localization of an immunoreactive domain of this protein. Furthermore, analogous to the plasmid-encoded borrelial outer surface proteins, the predicted surface-exposed portion of chromosomally encoded P66 appears to be antigenically heterogenous.

The flagellin genes from six Borrelia species were cloned, sequenced and characterized at the molecular level. The flagellin genes of two relapsing fever Borrelia species, B. hermsii and B. crocidurae, three Lyme disease genomic species, B. burgdorferi, B. afzelii and B. garinii, and the avian borreliosis agent, B. anserina, were compared and showed an 85-93% sequence identity to each other. Comparison of the fla genes from the different Lyme borreliosis spirochaetes revealed that they were 94-99% identical. Nucleotide sequencing of the fla gene and primer extension on isolated mRNA from both B. hermsii (as transcribed in Escherichia coli) and B. burgdorferi (as transcribed in the natural host) identified the putative transcriptional start points, the ribosomebinding sites and the promoter regions of these genes. The deduced promoter of the Borrelia flagellin gene resembled neither the sigma 70 promoter of prokaryotes, as seen for the genes for the outer-surface proteins A and B in Lyme disease Borrelia and the genes for the variable major proteins 7 and 21 of B. hermsii, nor the sigma 28 consensus promoter region of motility genes from other bacteria. Instead, the promoter of the fla gene in Borrelia has most similarity to the bacteriophage SP01 sigma gp33-34 promoter sequence of Bacillus subtilis.

The lipidated major outer surface protein, OspA, of the Lyme disease spirochaete may be important in the pathogenesis during Lyme borreliosis. To produce sufficient amounts of purified OspA variants to perform pathogenesis studies in vivo and in vitro, different recombinant OspA expression systems in Escherichia coli were constructed. Recombinant OspA variants were produced as a full-length molecule, as a truncated variant lacking the N-terminal lipidated cysteine, or as a fusion protein with the synthetic dimer of Staphylococcus aureus protein A IgG binding domain (ZZ). In order to produce the fall-length protein, four different promoters were evaluated. These were combined with either the OspA original signal sequence or the E. coli Brauns lipoprotein signal sequence, lpp. The most efficient production of the full-length lipidated OspA was mediated by the constitutive β-lactamase promoter in combination with lipoprotein signal sequences. For production of truncated nonlipidated OspA the S. aureus protein A signal sequence was ligated to the OspA open reading frame. Alternatively, truncated OspA was produced intracellularly using expression vectors that lack signal sequences. Production of nonlipidated protein with at heterologous signal peptide resulted in a soluble protein located mainly in the periplasm and in the culture medium. The full-length lipidated OspA, on the other hand, was associated mainly with the membrane fraction. The production level of the lipidated recombinant OspA was much lower than the level obtained with the truncated ZZ-OspA fusion protein.

A 66-kDa protein (p66) associated with the outer membrane of Lyme disease Borrelia was analysed at the molecular level. The chromosomal genes encoding p66 in B. burgdorferi B31, B. afzelii ACAI, and B. garinii Ip90 were sequenced. Database searches revealed that the p66 gene sequences were homologous to a previously reported gene fragment of unknown function. The deduced amino acid sequences of p66 in different Lyme disease borreliae were 92–94% identical and had no homologs in the databases. Proteolytic cleavage patterns of p66 and a computer-predicted single trans-membrane helix suggested the presence of surface-exposed epitopes on the C-terminus.

THE Lyme disease spirochaete, Borrelia burgdorferi s.l. , is the only Borrelia known to infect both mammals and birds1. The main vertebrate reservoirs of B. burgdorferi are thought to be various small and intermediate size mammals2, but the importance of birds as a reservoir has not been thoroughly explored. In the Northern and Southern Hemispheres the seabird tick, Ixodes uriae, is prevalent and closely associated with many species of colony-nesting marine birds3. Here we report the presence of spirochaetes, demonstrated by immunofluorescent assay, by polymerase chain reaction and in culture, in I. uriae infesting razorbills on an island in the Baltic Sea. This island is free from mammals. The protein profile of the spirochaetes and the sequences of their flagellin and ospA genes are identical to those of the Lyme disease spirochaete, Borrelia burgdorferi s.l. , previously isolated from I. ricinus on a nearby island. In biopsies from the foot web of razorbills, B. burgdorferi-specific DNA was detected after amplification by polymerase chain reaction. Our results suggest that birds play an important part in the maintenance of B. burgdorferi and that mammals may not be a prerequisite for its life cycle.

Biochemical and immunochemical studies of the outer membrane proteins of Borrelia burgdorferi have shown that the OspA and OspB proteins from strains of different geographic origins may differ considerably in their reactivities with monoclonal antibodies and in their apparent molecular weights. To further characterize this variation in Osp proteins between strains, the osp operons and deduced translation products from two strains, one from Sweden (ACAI) and one from eastern Russia (Ip90), were studied. Polyacrylamide gel electrophoresis and Western blot (immunoblot) analyses confirmed differences between ACAI, Ip90, and the North American strain B31 in their Osp proteins. The sequences of the ospA and ospB genes of ACAI and Ip90 were compared with that of the previously studied osp operon of B31 (S. Bergström, V. G. Bundoc, and A. G. Barbour, Mol. Microbiol. 3:479-486, 1989). The osp genes of ACAI and Ip90, like the corresponding genes of B31, were found on plasmids with apparent sizes of about 50 kb and are cotranscribed as a single unit. Pairwise comparisons of the nucleotide sequences revealed that the ospA genes of ACAI and Ip90 were 85 and 86% identical, respectively, to the ospA gene of strain B31 and 86% identical to each other. The ospB sequences of these two strains were 79% identical to the ospB gene of B31 and 81% identical to each other. There was significantly greater similarity between the ospA genes of the three different strains than there was between the ospA and ospB genes within each strain. These studies suggest that the duplication of osp genes in B. burgdorferi occurred before the geographical dispersion of strains represented by ACAI, Ip90, and B31.