Figure 2. Unrooted phylogenetic trees showing relationship of Cooleyi genotype to other rickettsiae. Scale bar represents genetic distance of 5% by using the Jukes-Cantor formula. Bootstrap values were derived by using the Fitch algorithm. Circled bootstrap values are for nodes indicated by the dashed line. Dotted lines show the actual position of some closely related spotted fever group species. A. 17 kDa. B. gltA. C. rompA. A 10-μl aliquot of phenol:chloroform-extracted DNA was used as a template to amplify portions of four rickettsial genes: 17 kDa protein gene, gltA (citrate synthase), rickettsial outer membrane protein A (rompA), and rickettsial outer membrane protein B (rompB). In all amplification reactions, R. rickettsii DNA was used as a positive control. A 434 bp-portion of the Rickettsia genus-specific 17 kDa protein was amplified (16). A 381 bp-portion of the rickettsial gltA gene was amplified from extracted DNA by using primer pairs Rp CS.877p and Rp CS.1258n (17). A 532 bp-portion of the rickettsial rompA gene was amplified from extracted tick DNA by using primer pairs Rr190.70n and Rr190.602p (17). Primer pair BG1-21 and BG2-20 (BG-12) was used in a polymerase chain reaction (PCR) for the amplification of a 650 bp-portion of the rompB gene (18). GenBank accession numbers for the portions of the I. scapularis rickettsia 17 kDa, rompA, and gltA gene sequences are AFO31534, AFO31535, and AFO31536, respectively. GenBank accession numbers for the available 17 kDa gene sequences included in phylogenetic analyses are R. canada, M82879; R. felis, M82878; R. prowazekii, M28482; R. typhi, M28481; AB Bacterium, U04162; R. amblyommii, U11013; R. japonica, D16515; R. honei, M99391; R. rickettsii, M16486; R. conorii, M28480; R. montana, U11017; R. rhipicephali, U11020; R. parkeri, U17008; and R. australis, M74042. GenBank accession numbers for the available gltA sequences included in phylogenetic analyses are AB Bacterium, U20242; R. bellii, U59716; R. typhi, U20245; R. prowazekii, U20244; R. montana, U74756; R. slovaca, U59725; R. conorii, U20243; R. sibirica, U59734; R. africae, U59733; Thai tick typhus rickettsia, U59726; R. rickettsii, U59729; R. honei, AFO22817; R. parkeri, U59732; R. japonica, U59724; R. massiliae, U59719; R. rhipicephali, U59721; R. aeschlimanni, U74757; R. felis, U33922; R. australis, U59718; R. akari, U59717; R. canada, U20241; and R. helvetica, U59723. GenBank accession numbers for the rompA sequences included in phylogenetic analyses are R. massiliae, U43793; R. rhipicephali, U43803; R. aeschlimanni, U43800; R. conorii, U43794; Israeli tick typhus rickettsia, U43797; R. peacockii, U55821; R. slovaca, U43808; R. parkeri, U43802; R. africae, U43790; R. sibirica, U43807; Thai tick typhus rickettsia, U43809; R. rickettsii, U55822; R. japonica, U43795; and R. montana, U43801.
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