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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.

Polymerase chain reaction primers specific for Ehrlichia chaffeensis were used to amplify DNA from extracts of pooled ticks. Amplification was performed on extracts from 140 pools (1,579 total ticks) consisting of three tick genera collected from five states. The characteristic 389-basepair product was observed after amplification of extracts from seven different pools of adult Amblyomma americanum (117 pools, 1,462 ticks), but not from pools of nymphs. No specific product was observed after amplification of 20 pools (105 ticks) of Dermacentor variabilis and three pools of Ixodes scapularis (12 ticks). Ehrlichia chaffeensis was present in A. americanum at a minimum frequency of > or = 0.48%, suggesting that A. americanum may be a vector of human ehrlichiosis.

We developed a polymerase chain reaction (PCR) that specifically amplifies a fragment of the flagellin gene (fla) of Borrelia burgdorferi, the causative agent of Lyme disease. This fla target, amplified with nested primers, was conserved among all 80 strains of B. burgdorferi tested. Strains examined included cultures from ticks, humans, and rodents from major B. burgdorferi-endemic regions of the United States and parts of Europe and Asia. Templates from B. hermsii, B. parkeri, B. turicatae, and B. coriaceae were not amplified, nor were eukaryotic DNAs from three tick genera. Several host DNAs potentially present in a tick blood meal also were not amplified. Approximately six B. burgdorferi per PCR reaction could be detected by ethidium bromide staining of amplified DNA. Colony-raised Ixodes dammini were used to evaluate the method. One infected nymph in a pool of 40 ticks was routinely detected. The specificity of the assay for detecting B. burgdorferi-infected ticks in pools was 94% (29 of 31). This protocol should prove useful for assessing infection rates in other putative arthropod vectors.