Approximately 118Borreliaisolates were cultured from a variety of rodents, birds, and ticks collected in the southern United States. In addition to a highly diverse group ofBorrelia bissettiistrains and a homogenous group ofBorrelia burgdorferisensu stricto strains, a group of 16 isolates with unusual characteristics was found. The isolates were cultured from ear biopsy samples of the rodentsPeromyscus gossypinusandNeotoma floridanatrapped at five localities in South Carolina. A multilocus sequence analysis of therrf-rrlintergenic spacer, 16S rRNA,fla,ospA, andp66genes were used to clarify the taxonomic status of the new group ofB. burgdorferisensu lato isolates. Thirteen species of theB. burgdorferisensu lato complex were used as controls. Unique restriction fragment length polymorphism patterns of therrf-rrlintergenic spacer region andflagene were recognized. Unique signature nucleotides were also found in the 16S rRNA gene. A phylogenetic analysis shows that the 16 new isolates cluster together but separately from the other species in theB. burgdorferisensu lato complex. Our data strongly support the recognition of the 16 isolates as a newB. burgdorferisensu lato species. We propose to name this genospecies “Borrelia carolinensis” with respect to the place of its currently known geographic location.

Ixodes ricinus L. is the principal European vector of Borrelia burgdorferi sensu lato, the causative agent of Lyme borreliosis. Subtractive hybridization was used to isolate tick genes that were induced in whole ticks after blood meals on uninfected and B. burgdorferi-infected guinea pigs. Novel cDNA clones with similarity to cytochrome c oxidase, salivary secreted protein, actin, and a cysteine protease propeptide were induced after a blood meal. Novel cDNA clones with similarity to thioredoxin peroxidases, dolichyl-phosphate β-glucosyltransferase, glutathione S-transferase, defensin, ML domain-containing protein, and von Willebrand factor were induced after B. burgdorferi infection. Virtual Northern analysis was used to verify that these genes were differentially expressed in ticks after a pathogen-infected blood meal and to detect their tissues of expression. The characterization of genes that are induced after an infected blood meal is essential for gaining an understanding of the molecular mechanisms that underlie vector–pathogen interactions.

Analysis of borrelia isolates collected from ticks, birds, and rodents from the southeastern United States revealed the presence of well-established populations of Borrelia burgdorferi sensu stricto, Borrelia bissettii , Borrelia carolinensis , and Borrelia sp. nov. Multilocus sequence analysis of five genomic loci from seven samples representing Borrelia sp. nov. isolated from nymphal Ixodes minor collected in South Carolina showed their close relatedness to California strains known as genomospecies 1 and separation from any other known species of the B. burgdorferi sensu lato complex. One nucleotide difference in the size of the 5S-23S intergenic spacer region, one substitution in 16S rRNA gene signature nucleotides, and silent nucleotide substitutions in sequences of the gene encoding flagellin and the gene p66 clearly separate Borrelia sp. nov. isolates from South Carolina into two subgroups. The sequences of isolates of each subgroup share the same restriction fragment length polymorphism patterns of the 5S-23S intergenic spacer region and contain unique signature nucleotides in the 16S rRNA gene. We propose that seven Borrelia sp. nov. isolates from South Carolina and two California isolates designated as genomospecies 1 comprise a single species, which we name Borrelia americana sp. nov. The currently recognized geographic distribution of B. americana is South Carolina and California. All strains are associated with Ixodes pacificus or Ixodes minor and their rodent and bird hosts.

Molecular analysis of a clinical sample confirmed the presence of Borrelia bissettii DNA in cardiac valve tissue from a patient with endocarditis and aortic valve stenosis. This evidence strongly supports the involvement of B. bissettii in Lyme disease in Europe.

Until recently, three spirochete genospecies were considered to be the causative agents of Lyme borreliosis (LB) in Europe: Borrelia burgdorferi sensu