Population genetics and phylogenetic analysis of Colorado Borrelia burgdorferi.

Borrelia burgdorferiis transmitted in an enzootic cycle in Colorado between the tick Ixodes spinipalpis and the woodrat Neotoma mexicana. The genetic relationship of Colorado isolates to other B. burgdorferi isolates is unknown nor have relationships among various Colorado isolates been determined. Portions of the flagellin ( fla ), 66- kD protein, and outer surface protein A (ospA) genes were amplified from 71 Colorado isolates, screened for genetic variability using single strand conformation polymorphism analysis, and unique alleles were sequenced. Colorado isolates were most similar to tick isolates from California and New York isolate 25015. Genetic distances among Colorado ospA sequences were the same or higher than distances among other isolates whereas distances among fla sequences tended to be the same or lower. The index of association (I A ) was calculated among all loci as a measure of clonality. The IA among Colorado isolates was similar to IA previously estimated among other United States isolates. Borrelia burgdorferi sensu lato is a genetically diverse species complex consisting of four genospecies. 1-6 A gen- ospecies is a bacterial subspecies defined by degree of DNA relatedness as assessed by nucleotide sequence data or DNA- DNA reassociation. Three of these genospecies, B. burgdor- feri ss, B. garinii, and B. afzelii, cause Lyme disease and related disorders in humans and are distributed throughout Europe and Eurasia. Borrelia japonica, the fourth genospe- cies, appears to be nonpathogenic and is restricted to rodents and ticks in Asia. 2, 5 Borrelia burgdorferi ss is maintained in a diversity of enzootic cycles in the United States involving ticks of the genus Ixodes and rodent or lagomorph reser- voirs. 7-10 In California where indigenous cases of Lyme dis- ease occur, Ixodes pacificusacts as a vector of B. burgdorferi to humans 11 and is maintained in enzootic cycles primarily involving the dusky-footed wood rat (Neotoma fucipes). 7, 12 10 No known 13 Colorado isolates were identified phylogenetically as B. burgdorferi ss. We demonstrated large difference in the frequencies and diver- sity of alleles in cultured spirochetes as compared with those that were amplified from infected ticks. This observation suggested that culturing in Barbour-Stoenner-Kelley-H (BSK)-H medium selects for specific B. burgdorferi geno- types in Colorado and may underestimate the diversity of genotypes in this and potentially other enzootic cycles. Of greater concern, these results suggest the possibility that the prevalence of B. burgdorferi may be underestimated in cer- tain enzootic cycles in the United States if they also involve genotypes that cannot be cultured. The primary goal of this study was to perform a phylogenetic analysis of each of the alleles at the three loci to estimate the relationship of Col- orado isolates relative to other B. burgdorferi ss isolates. In addition, we compare genetic diversity among Colorado iso- lates relative to the diversity among other B. burgdorferi isolates. The rate with which selection can act on a set of organisms is proportional to the genetic variance among those organisms.