Phylogenetic analysis of the spirochetes Borrelia parkeri and Borrelia turicatae and the potential for tick-borne relapsing fever in Florida

Abstract

Isolates of Borrelia turicatae, Borrelia parkeri, and the Florida canine borrelia (FCB) were examined to further phylogenetically characterize the identities of these spirochetes in the United States. DNA sequences of four chromosomal loci (the 16S rRNA gene, flaB, gyrB, and glpQ) were determined for eight isolates of B. turicatae and six isolates of B. parkeri, which grouped the spirochetes into two distinct but closely related taxa (>98% sequence identity) separate from Borrelia hermsii. The FCB was clearly separated with the group identified as B. turicatae, confirming this bacterium as a relapsing fever spirochete. Therefore, the potential for tick-borne relapsing fever in humans and other animals exists in Florida and future efforts are needed to determine the enzootic hosts and distribution of this spirochete in the southeastern United States. Analysis of plasmids demonstrated both linear and circular forms in B. turicatae but only linear plasmids in B. parkeri, which should be of interest to investigators concerned with plasmid diversity and evolution within this group of spirochetes.

FIG. 1.

Protein profiles of whole-cell lysates of B. turicatae and B. parkeri (A), immunoblot reactivity with Borrelia genus-specific monoclonal antibody H9724 (B), and lack of reactivity with B. hermsii-specific monoclonal antibody H9826 (C). The arrow indicates the position in the stained gel of flagellin, to which the monoclonal antibodies bind. B. hermsii DAH is the only lysate recognized by H9826. Molecular mass standards (MMS) are shown at the left (sizes are in kilodaltons).

FIG. 2.

Phylogenetic tree of the concatenated DNA sequences for the four loci in B. turicatae and B. parkeri with B. hermsii DAH used for the outgroup. The tree was constructed with ClustalV and the neighbor-joining method to distinguish the two species. The reliability of the tree topology was gauged by performing bootstrap analysis using 1,000 replicates. Bootstrap values of 100% at nodes, where the configuration to the right was found, are shown. The bar shown in the graph provides scale to the branch lengths and represents the number of substitutions per site (0.001). The FCB clearly falls within the B. turicatae cluster.

FIG. 3.

Reverse-field agarose gel of total genomic DNA for plasmids in B. turicatae and B. parkeri (A) and Southern blot assay with the blyA probe to identify the approximately 30-kb circular plasmids (B). Arrows on the left indicate the positions of the aberrantly migrating 30-kb circular plasmids in B. turicatae and B. hermsii that hybridized with the probe but were not present in B. parkeri. Positions of molecular size standards (MSS) are shown on the left (sizes are in kilobases).

FIG. 4.

Two-dimensional agarose gel electrophoresis to determine the presence of circular plasmids in several species of Borrelia (A) and Southern blot assays utilizing the same gel samples transferred and hybridized with the blyA probe (B) and the bdrC probe (C). Arrows on the left identify the circular plasmids with retarded migration in the second dimension that hybridized with both probes. Two samples of the same isolate of B. turicatae were included before (Low) and after (High) 1 year of continuous in vitro cultivation.