(the Gulf Coast tick) an aggressive human-biting Nearctic and Neotropical tick

(the Gulf Coast tick) an aggressive human-biting Nearctic and Neotropical tick is the principal vector of in the United States. human-biting Nearctic and Neotropical tick that is distributed widely across many countries in the Western Hemisphere. In the United States is the principal vector of occur throughout the southeastern and south-central says and along much of the eastern seaboard. Molecular surveys of Gulf Coast ticks collected from several locations in multiple says within its southern and eastern range reveal estimated rates of contamination with in 8-52% of questing adult Bleomycin sulfate ticks (Sumner et al. 2007 Paddock et al. 2010 Fornadel et al. 2011 Wright et al. 2011 Varela-Stokes et al. 2011 Jiang Bleomycin sulfate et al. 2012 Ferrari et al. 2012 Nadolny et al. 2014 Florin et al. 2013 Pagac et al. Bleomycin sulfate 2014 and Florin et al. 2014 More than 35 cases of rickettsiosis have been recognized in patients from 9 says (Paddock and Goddard 2015 “Rickettsiae andeanae” was first explained from specimens of and collected in Peru (Blair et al. 2004 and subsequently from Gulf Coast ticks in the United States (Paddock et al. 2010 and other tick species in Argentina Brazil and Chile (Pacheco et al. 2007 Abaraca et al. 2012 and Nieri-Bastos et al. 2014 “R. andeanae” has been isolated recently in culture although some difficulties remain in establishing continuously infected cell lines (Luce-Fedrow et al. 2012 and Ferrari et al. 2013 To our knowledge no cases of rickettsiosis have been explained from Kansas or Oklahoma despite well-established populations of in those says which have existed for more than 40 years. In this study we used molecular techniques to evaluate adult Gulf Coast ticks collected from multiple sites in Kansas and Oklahoma for infections with or “ticks were collected from vegetation by using fabric drags or flags at multiple sites in 9 counties of Kansas (Anderson Butler Crawford Geary Morris Neosho Osage Riley and Shawnee) and 9 counties of Oklahoma (Cleveland Cotton Kiowa Lincoln Payne Osage Tillman Tulsa and Washington). Field-collected specimens were placed in 70% ethanol and Lep transported to the laboratory where these were air-dried recognized using a standard taxonomic important (Keirans and Litwak 1989 transferred to individual 1.5 ml microcentrifuge tubes and stored at ?80 °C prior to DNA extraction. Molecular analyses Genomic DNA was extracted from tick specimens by using a DNA Minikit (Qiagen Valencia CA) and eluted in a final volume of Bleomycin sulfate 100 μL. Extracted samples were evaluated for DNA of and “gene (Jiang et al. 2012 For each real-time PCR assay 2.5 μL of tick extract was mixed with 0.4 μM of the forward and reverse primers (Rpa129F and Rpa224R for R. andeane”) in a final reaction volume of 25 μL. Cycling was performed on an Mx3005P thermal cycler (Agilent Santa Clara CA) and conditions consisted of 15 min at 95 °C 45 cycles of 1 1 min at 95 °C and 1 min at 60 °C. Ct values <40 were considered positive for the respective agent. Both assays were validated by screening a panel of DNA Bleomycin sulfate extracts of ticks naturally infected with or “PCR assay and sequence analysis (Sumner et al. 2007 and Paddock et al. 2010 or recognized unfavorable for DNA of and “species real-time PCR assay targeting sequence of the R andeanae” by the real-time assay were evaluated by using a hemi-nested PCR assay with primers RR190.70 and RR190.701 in the primary reaction and RR190.70 and RR190.602 in the secondary reaction (Sumner et al. 2007 followed by sequence analysis of the amplified segments of the rickettsial gene. A subset of 10 extracts representing ticks collected from 5 counties in Kansas and 10 extracts representing ticks collected from 5 counties in Oklahoma were selected for further analysis to verify the morphological species identification by using a standard PCR assay with primers T1B and T2A (Beati and Keirans 2001 and sequencing of the amplified segments of the ixodid mitochondrial 12S ribosomal DNA gene. RESULTS A total of 216 adult Gulf Coast ticks were evaluated comprising 53 male and 41 female specimens from Kansas collected during 2012-2013 and 52 male and 70 female specimens collected from Oklahoma during 2011-2014 (Table 1). Of the Kansas specimens 51 (54%) were obtained from multiple sites in Geary County during May-July 2013 whereas 87 (71%) of the total Oklahoma specimens were collected from 3 sites in Payne county during 2011-2013 (Fig. 1). Of the 20 tick extracts.