PCR-Reverse Line Blot Typing Method Underscores the Genomic Heterogeneity of Borrelia valaisiana Species and Suggests Its Potential Involvement in Lyme Disease

Abstract

Detection of the Borrelia burgdorferi sensu lato complex in biological samples is currently done by conventional immunological and molecular biological methods. To improve on the accuracy of these methods and to simplify the procedure for testing large numbers of samples, a solid-phase sandwich hybridization system readily applicable to the detection of PCR products has been designed. This colorimetric detection system relies on the use of polybiotinylated detection probes and of specific capture oligonucleotides covalently linked at allocated positions on nylon membrane strips. From a phylogenetic analysis on a great number of ospA gene sequences, we have designed and synthesized a set of PCR primers specific to the five Borrelia burgdorferi sensu lato genospecies present in Europe and a subset of probes (capture and detection probes) specific to these five genospecies (B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. valaisiana, and B. lusitaniae). This combined PCR hybridization system was evaluated with a large number of various B. burgdorferi isolates and clinical specimens. These analyses clearly showed that the system could be used as a typing method to distinguish five genospecies belonging to the B. burgdorferi sensu lato complex. In addition, the study showed that B. valaisiana strains might be more heterologous than suspected up to now and clustered into three genomic groups.

Characterization of Borrelia lusitaniae Isolates Collected in Tunisia and Morocco

“…The relative level of conservation of ospA sequences from B. lusitaniae could be attributed to the restricted geographic extension and to the interaction with only one vector species and possibly one predominant reservoir host. This fact contrasts with the diversity reported for B. valaisiana, which is more widely distributed and is associated with several tick species, requiring adaptation to a broad range of vectors (12,22,24,32,40). Our analysis of the restriction polymorphism of the small rrf-rrl spacer emphasized a high monomorphism that was confirmed by the conservation of sequences within B. lusitaniae isolates from North Africa.…”

“…Amplification products from the ospA gene were sequenced using the BigDye Terminator cycle sequencing kit (Applied Biosystems) as described in reference 12. Primers used in this study were direct and reverse primers 1 and 2, as previously described (12).…”

“…The genetic diversity of pathogenic species has been largely investigated both at the species level and at the strain level within each species (41). Otherwise, although its pathogenicity for humans is only suspected (31, 33), Borrelia valaisiana represents a widely distributed species in ticks throughout Europe and Asia and therefore has been extensively studied (12,22,32,38,40).In contrast to these species, little data are available concerning Borrelia lusitaniae. Only sporadic isolates have been characterized from ticks collected in the Czech Republic, Moldavia, Ukraine, and Belarus (20), in Slovakia (11), in Spain (3), in Poland (25), in France (30), in Switzerland (15-17), or in Turkey (13), and these isolates did not show any significant diversity.…”

“…The genetic diversity of pathogenic species has been largely investigated both at the species level and at the strain level within each species (41). Otherwise, although its pathogenicity for humans is only suspected (31, 33), Borrelia valaisiana represents a widely distributed species in ticks throughout Europe and Asia and therefore has been extensively studied (12,22,32,38,40).…”

Borrelia carolinensissp. nov., a New (14th) Member of theBorrelia burgdorferiSensu Lato Complex from the Southeastern Region of the United States

“…Alignment of the partial sequences of the B. carolinensis ospA gene with two oligonucleotide probes, designed for the detec- tion of B. burgdorferi sensu lato species (Table 2), revealed 100% identity within two different regions of the gene. The clustering of Borrelia strains in the phylogenetic tree based on ospA DNA sequences is in agreement with the classification based on the sequence analysis of conserved chromosomal genes, as well as with data obtained by pulsed-field gel electrophoresis and randomly amplified polymorphic DNA fingerprinting (2,12). Analysis of the p66 gene.…”