Phylogenetic Analysis of Borrelia Species Based on Flagellin Gene Sequences and Its Application for Molecular Typing of Lyme Disease Borreliae

Abstract

We determined almost complete flagellin gene sequences of various Borrelia species and aligned them with previously published sequences. A neighbor-joining phylogenetic analysis showed that the genus Borrelia was divided into the following three major clusters: New World relapsing fever borreliae (BorreZia turicatae, Burreliu parkeri, and Borrelia hermsii), Old World relapsing fever borreliae (Borrelia crocidurae, Borrelia duttonii, and Borrelia hispanica), and Lyme disease borreliae (Borrelia burgdurferi sensu stricto, Borrelia garinii, and Borrelia afzelii). Agents of animal spirochetosis (Borrelia coriaceae and Borrelia anserina) and species of unknown pathogenicity (Borrelia rniyamotoi and Borrelia Zonestari) were related to relapsing fever borreliae. Although the Lyme disease borreliae, two related species (Borrelia japonica and Borrelia andersonii), and some newly described genomic groups (groups PotiB2, VS116, DN127, Hk501, and Ya501) were closely related to each other, each taxon formed an independent branch on the phylogenetic tree. The data obtained in this study indicate that the flagellin genes are useful in Borrelia taxonomy. To distinguish the Lyme disease borreliae from related organisms easily, we designed an oligonucleotide primer set for the flagellin gene and performed a PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. The primer set amplified an approximately 580-bp DNA fragment that included species-specific restriction sites, and HapII, HhaI, CelII, HincII, or DdeI digestion of the product resulted in distinctively different PCR-RFLP patterns. The PCR-RFLP typing method which we developed should facilitate rapid identification of Lyme disease borreliae and related organisms obtained from biological and clinical specimens.

Delineation of a New Species of the Borrelia burgdorferi Sensu Lato Complex, Borrelia americana sp. nov

“…Strains SCW-30e and SCW-30g were 100% identical to each other and differ from the rest of the B. americana strains by having isoleucine instead of methionine at amino acid position 160. The DdeI RFLP pattern of the B. americana fla gene is unique and has not been described previously (13). Restriction endonucleases HapII, HhaI, HincII, and CelII had no sites on the fla amplicon of B. americana.…”

Typing African Relapsing Fever Spirochetes

“…Many widely used techniques for molecular typing have been applied to Borrelia with variable success ( 5 , 6 ). Of concern when these methods are used for characterizing these spirochetes is the organisms' ability to undergo multiphasic antigenic variation, which can be associated with large inter- or intraplasmidic recombinations/duplications ( 7 , 8 ).…”

“…Others have used the flagellin gene as a target for population structure ( 5 , 13 ); however, as with 16SRNA, these methods, although useful for interspecies comparisons, are of limited value for discrimination of African relapsing fever spirochetes and for intraspecies analysis. A partial intergenic spacer region (IGS; rrs [16S rRNA]- ileT [tRNA]) has recently been used for typing Lyme-associated Borrelia spp., together with other genetic loci ( 14 ).…”

Glycerophosphodiester Phosphodiesterase Gene ( glpQ ) of Borrelia lonestari Identified as a Target for Differentiating Borrelia Species Associated with Hard Ticks (Acari:Ixodidae)

“…Evidence suggests that persons in these regions bitten by the hard tick, Amblyomma americanum, who develop a red, expanding rash with central clearing (indistinguishable from erythema migrans, the hallmark rash of LD) are infected with a spirochete named Borrelia lonestari (3,4,8,18 (14). With these data, and given that B. lonestari is more closely related to RFG spirochetes than to LD spirochetes based on 16S ribosomal DNA (rDNA) and fla analyses (3,4,7,8,13), we decided to look for an ortholog of glpQ in B. lonestari-positive tick DNA. The 16S rRNA and fla genes are highly conserved and are present in all Borrelia species, making them ill suited for development of rapid, differential diagnostic assays.…”