Genetic Heterogeneity ofBorrelia burgdorferiSensu Lato in the Southern United States Based on Restriction Fragment Length Polymorphism and Sequence Analysis

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Abstract
Fifty-six strains ofBorrelia burgdorferisensu lato, isolated from ticks and vertebrate animals in Missouri, South Carolina, Georgia, Florida, and Texas, were identified and characterized by PCR-restriction fragment length polymorphism (RFLP) analysis ofrrf(5S)-rrl(23S) intergenic spacer amplicons. A total of 241 to 258 bp of intergenic spacers between tandemly duplicatedrrf(5S) andrrl(23S) was amplified by PCR.MseI andDraI restriction fragment polymorphisms were used to analyze these strains. PCR-RFLP analysis results indicated that the strains represented at least three genospecies and 10 different restriction patterns. Most of the strains isolated from the tickIxodes dentatusin Missouri and Georgia belonged to the genospeciesBorrelia andersonii. Excluding theI. dentatusstrains, most southern strains, isolated from the ticksIxodes scapularisandIxodes affinis, the cotton rat (Sigmodon hispidus), and cotton mouse (Peromyscus gossypinus) in Georgia and Florida, belonged toBorrelia burgdorferisensu stricto. Seven strains, isolated fromIxodes minor,the wood rat (Neotoma floridana), the cotton rat, and the cotton mouse in South Carolina and Florida, belonged toBorrelia bissettii. Two strains, MI-8 from Florida and TXW-1 from Texas, exhibitedMseI andDraI restriction patterns different from those of previously reported genospecies. Eight Missouri tick strains (MOK-3a group) hadMseI patterns similar to that ofB. andersoniireference strain 21038 but had aDraI restriction site in the spacer. Strain SCGT-8a hadDraI restriction patterns identical to that of strain 25015 (B. bissettii) but differed from strain 25015 in itsMseI restriction pattern. Strain AI-1 had the sameDraI pattern as other southern strains in theB. bissettiigenospecies but had a distinctMseI profile. The taxonomic status of these atypical strains needs to be further evaluated. To clarify the taxonomic positions of these atypicalBorreliastrains, the complete sequences ofrrf-rrlintergenic spacers from 20 southeastern and Missouri strains were determined. The evolutionary and phylogenetic relationships of these strains were compared with those of the described genospecies in theB. burgdorferisensu lato species complex. The 20 strains clustered into five separate lineages on the basis of sequence analysis. MI-8 and TXW-1 appeared to belong to two different undescribed genospecies, although TXW-1 was closely related toBorrelia garinii. The MOK-3a group separated into a distinct deep branch in theB. andersoniilineage. PCR-RFLP analysis results and the results of sequence analyses of therrf-rrlintergenic spacer confirm that greater genetic heterogeneity exists amongB. burgdorferisensu lato strains isolated from the southern United States than among strains isolated from the northern United States. TheB. andersoniigenospecies and its MOK-3a subgroup are associated with theI. dentatus-cottontail rabbit enzootic cycle, butI. scapulariswas also found to harbor a strain of this genospecies. Strains that appear to beB. bissettiiin our study were isolated fromI. minorand the cotton mouse, cotton rat, and wood rat. TheB. burgdorferisensu stricto strains from the south are genetically and phenotypically similar to the B31 reference strain.