Comparative analysis of the Borrelia garinii genome.

Three members of the genus Borrelia (B.burgdorferi, B.garinii, B.afzelii) cause tick-borne borreliosis. Depending on the Borrelia species involved, the borreliosis differs in its clinical symptoms. Comparative genomics opens up a way to elucidate the underlying differences in Borrelia species. We analysed a low redundancy whole-genome shotgun (WGS) assembly of a B.garinii strain isolated from a patient with neuroborreliosis in comparison to the B.burgdorferi genome. This analysis reveals that most of the chromosome is conserved (92.7% identity on DNA as well as on amino acid level) in the two species, and no chromosomal rearrangement or larger insertions/deletions could be observed. Furthermore, two collinear plasmids (lp54 and cp26) seem to belong to the basic genome inventory of Borrelia species. These three collinear parts of the Borrelia genome encode 861 genes, which are orthologous in the two species examined. The majority of the genetic information of the other plasmids of B.burgdorferii is also present in B.garinii although orthology is not easy to define due to a high redundancy of the plasmid fraction. Yet, we did not find counterparts of the B.burgdorferi plasmids lp36 and lp38 or their respective gene repertoire in the B.garinii genome. Thus, phenotypic differences between the two species could be attributable to the presence or absence of these two plasmids as well as to the potentially positively selected genes.

Differential expression of outer surface proteins A and C by individual Borrelia burgdorferi in different genospecies.

Previous studies with different Borrelia burgdorferi sensu stricto (s.s.) strains revealed that temperature as well as cocultivation with tick cells modulates the expression of outer surface proteins (Osp) A and C. We investigated the effects of temperature and of interaction with tick cells in culture on the expression of OspA and OspC of the B. afzelii clones cPKo97 and cPKo345 in comparison to the B. burgdorferi s.s. strain N40. To follow the dynamics of Osp expression of single borreliae we used indirect immunofluorescence microscopy with double staining of OspA and OspC. Clone PKo345 always showed expression of only OspA, regardless the conditions it was subjected to. Sequencing of the ospC gene disclosed a insertion leading to a stop codon after base 222 and inability to produce OspC. In cPKo97 and N40 OspC is down-regulated at lower temperatures and up-regulated at higher temperatures, which was especially pronounced on cocultivation with tick cells. Borreliae adherent to tick cells showed greater OspA expression compared to the nonadherent ones, an indication that OspA might play a role as adhesin for tick cells. Interestingly, cPKo97 and N40 displayed different patterns of Osp expression: cPKo97 simultaneously presents OspA and OspC on single borreliae, while N40 has either OspA or OspC on single cells. Adaptation of OspC expression in cPKo97 seems to occur by up- or down-regulation of this protein on single borreliae, as shown by alternating intensities of OspC expression at different temperatures. In contrast, N40 seem to consist of two subsets of borreliae one expressing only OspA and the other only OspC, and change in temperature results in growth benefit for one of these subtypes. Our findings indicate that, regarding OspA and OspC expression, response to temperature and cocultivation with tick cells of B. afzelii is comparable to B. burgdorferi s.s., but the mode of regulation seems phenotypically different. Further European isolates should be investigated for OspA and OspC regulation, especially in the face of vaccine development for the European situation.

An ospA-polymerase chain reaction/restriction fragment length polymorphism-based method for sensitive detection and reliable differentiation of all European Borrelia burgdorferi sensu lato species and OspA types.

We describe a sensitive and reliable method for detection and differentiation of the five relevant European Borrelia burgdorferi sensu lato species ( B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. valaisiana, and B. lusitaniae), based on a heminested ospA-PCR followed by restriction enzyme analysis. Sensitivity was one borrelia per PCR except for B. afzelii, where it was five per PCR. None of seven relapsing fever borreliae, eight Leptospira serovars or two Treponema species were amplified. Except B. garinii, each of the five B. burgdorferi s.l. species is represented by one or two characteristic restriction fragment length polymorphism (RFLP) patterns. Analysis of the heterogeneous group of B. garinii resulted in five different RFLP patterns, corresponding to the OspA types 3-7 associated with this species. In a pilot study on 529 Ixodes ricinus ticks from three different regions in Southern Germany, all species and OspA types were found except B. lusitaniae and B. garinii OspA type 7, arguing for a broad distribution of almost all OspA types. A further notable finding was the focal prevalence of OspA type 4, which has rarely been detected in ticks previously. Thus, the developed method provides a fast and simple tool for epidemiological studies on the heterogeneity of species and OspA types in Europe which has important implications for the development of vaccines and (microbiological) test systems for Europe.

Genetic analysis of Borrelia garinii OspA serotype 4 strains associated with neuroborreliosis: evidence for extensive genetic homogeneity.

Infection with Borrelia garinii outer surface protein (Osp) A serotype 4 strains has been correlated with the development of neuroborreliosis in Lyme borreliosis patients in Europe. OspA serotype 4 isolates have been recovered primarily from human cerebrospinal fluid, suggesting a tropism for this environment. Previous studies with monoclonal antibodies directed against OspA and OspC demonstrated that OspA serotype 4 strains are antigenically closely related. In view of the pronounced antigenic and genetic variability that has been noted in the Osps of other Borrelia isolates, we sought to determine if OspA serotype 4 strains represent a recently emerged clonal lineage of B. garinii. Toward this goal, a representative group of OspA serotype 4 strains was analyzed for traits that typically exhibit hypervariability among isolates that cause Lyme borreliosis. The following criteria were assessed: (i) ospC sequences, (ii) plasmid composition, (iii) genomic restriction fragment length polymorphism (RFLP) patterns, and (iv) the RFLP patterns of the upstream homology box (UHB) element which flanks members of the UHB gene family at their 5' end. Collectively, these analyses demonstrate genetic homogeneity, suggesting that OspA serotype 4 strains are a recently emerged clonal lineage with an apparent tropism for the central nervous system.

An improved recombinant IgG immunoblot for serodiagnosis of Lyme borreliosis.

We have previously described the use of the following recombinant antigens for serodiagnostic immunoblots: p83/100, p39, OspC and p41 (flagellin) internal fragment [Wilske et al. (1993) Med Microbiol Immunol 182:255-270; Rossler et al. (1997) J Clin Microbiol 35:2752-2758]. In our currently used immunoblot p83/100 is derived from strain PKo (Borrelia afzelii), p39 (BmpA) and OspC from strains PKa2 (B. hurgdorferi sensu stricto), PKo and PBi (B. garinii), respectively; the p41 (flagellin) internal fragments were cloned from strains PKo and PBi. In this study we describe the use of two additional recombinantly expressed highly immunogenic proteins Osp 7 (derived from PKo) and p58 (derived from PBi). A clinically well-defined panel of sera from 147 Lyme borreliosis patients and 139 controls previously tested by a standardized whole cell lysate immunoblot [Hauser et al. (1997) J Clin Microbiol 35:1433-1444] was investigated in the recombinant immunoblot without (old recombinant immunoblot) and with Ospl7 and p58 (new recombinant immunoblot) for IgG antibodies. The sensitivity of the recombinant IgG immunoblot for diagnosis of stage II and stage III could be significantly improved by addition of Osp17 and p58 without loss of specificity. With the exception of sera from patients with erythema migrans the diagnostic sensitivity is comparable to the whole cell lysate IgG immunoblot. The main advantage of the recombinant immunoblot is the easy identification of diagnostic bands, whereas the identification of bands in the whole cell lysate immunoblot is difficult. The recombinant immunoblot is especially suitable where large series of sera need to be investigated.

Osp17, a novel immunodominant outer surface protein of Borrelia afzelii: recombinant expression in Escherichia coli and its use as a diagnostic antigen for serodiagnosis of Lyme borreliosis.

Western blot analyses of the human humoral response of patients with Lyme borreliosis have shown that a 17-kDa protein is an immunodominant protein in late disease. Immune electron microscopy with a monoclonal antibody against this protein revealed that the 17-kDa protein is abundantly expressed on the surface of Borrelia afzelii strain PKo. Therefore, the protein has been renamed outer surface protein (Osp) 17. Recombinant Osp 17 of strain PKo was expressed in Escherichia coli and purified by chromatography. Immunoblot analysis of human sera showed a comparable sensitivity with recombinant and natural proteins. The DNA sequences of the osp17 genes from different B. afzelii strains were determined. The DNA sequences of the different osp 17 homologues (six isolates from skin, three isolates from CSF and one isolate from synovial fluid) had high sequence identities of at least 94%. Using a polyclonal antibody against recombinant Osp 17, it was shown that Osp 17 expression varied considerably among the investigated B. afzelii strains. As previously also observed for OspA- and OspC-encoding genes, the osp 17 gene is present in strains not expressing the respective protein. It has been shown that OspA and OspC expression varies in different environments such as tick and vertebrate host. Studies are underway to examine whether this is also true for Osp 17. For diagnostic purposes the use of recombinant Osp 17 has the advantage that the amount of Osp 17 antigen can be easily standardized for immunoblotting, and that this antigen can be used in a protein-specific enzyme-linked immunosorbent assay.