Questing ticks were collected during July 16–20, 2012, by using cloth drags, flags, and CO₂-baited traps, and by removing ticks from collectors (Table 1). Sites of collection were Fort Campbell (Christian County, Kentucky, and Montgomery County, Tennessee), Fort Knox (Bullitt, Hardin, and Meade Counties, Kentucky), and Wendell H. Ford Regional Training Center (WHFRTC; Muhlenberg County, Kentucky).

Multiple 2-person teams collected ticks during 15-minute periods; an average of 19 person-hours was spent sampling at each site. Target tick species were A. maculatum and Dermacentor variabilis, although A. americanum ticks were also collected. Human encounter rates (calculated by using all collection methods except CO₂-baited traps) for adult A. maculatum and D. variabilis ticks were ≈2 ticks/hour and 5 ticks/hour, respectively. No immature stages of these species were encountered. Field sites sampled were dominated by sericea (Lespedeza cuneata) and fescue (Festuca pratensis). Some adjacent areas had switchgrass (Panicum virgatum) and Indiangrass (Sorghastrum nutans). A. maculatum ticks appeared tolerant of exposed, unshaded sites and were often collected in the middle of these fields.

Ticks were identified by using the key of Keirans and Litwak (8). Specimens were individually placed in microcentrifuge tubes containing 500 μL of tissue lysis buffer (QIAGEN, Valencia, CA, USA) and 20 μL of proteinase K (QIAGEN), bisected with a sterile blade, and incubated at 56°C for ≥1 h. Nucleic acid was extracted by using the DNeasy Blood and Tissue Kit (QIAGEN).

Initial quantitative real-time PCRs (qPCRs) were performed by using the Rickettsia-specific Rick17b assay specific for the 17-kD antigen gene (4) and the LightCycler TaqMan Master (Roche Applied Sciences, Indianapolis, IN, USA) ready-to-use hot start reaction mixture in the LightCycler 2.0 instrument (Roche Applied Sciences). Final reactions contained 5 μL of template and 15 μL of master mixture. Master mixture contained 0.5 µmol/L primers, 0.4 μmol/L probe, LightCycler TaqMan Reaction Mixture (Roche Applied Sciences), and water. All qPCRs were performed at 95°C for 10 min and for 45 cycles at 95°C for 15 s and 60°C for 30 s.

Positive samples were further evaluated by using the SFG Rickettsia-specific conventional PCR with primer pair Rr190.70p and Rr190.602n, which is specific for the outer membrane protein A (ompA) gene of Rickettsia spp. and speciated by using PstI restriction fragment length polymorphism analysis (9). Identities of 9 positive samples were confirmed by sequencing a fragment of ompA (1,651 bp) or ompB (1,540 bp) genes (Table 2) (10). All A. maculatum tick samples positive for Rickettsia spp. were also tested for Candidatus Rickettsia andeanae by using the Rande qPCR (4).

A total of 404 adult ticks (105 A. maculatum and 299 D. variabilis) were collected and tested. Of these ticks, 3 A. maculatum and 44 D. variabilis ticks were collected from Fort Knox, 66 A. maculatum and 148 D. variabilis ticks were collected from Fort Campbell, and 36 A. maculatum and 107 D. variabilis ticks were collected from WHFRTC. Twenty-five (6.2%) of 404 ticks were infected with an SFG Rickettsia species. R. parkeri was detected in 15 (14.3%) of the A. maculatum ticks.

The ompA sequences (GenBank accession no. KJ741849) of A. maculatum ticks collected from Fort Campbell (n = 2) and WHFRTC (n = 2) were identical to those of R. parkeri strain Portsmouth (GenBank accession no. CP003341) and R. parkeri Maculatum 20 (GenBank accession no. U83449). R. montanensis was detected in 10 (3.3%) of the D. variabilis ticks; isolates from 5 tick samples were sequenced. The ompA sequences (GenBank accession no. KJ741850) of D. variabilis ticks from Fort Knox (n = 1) and WHFRTC (n = 2) were 99.9% identical with R. montanensis str. OSU 85–930 (GenBank accession no. CP003340). The ompB sequences (GenBank accession no. KJ741851) of 2 D. variabilis ticks collected at Fort Campbell were 99.9% identical with those of R. montanensis str. OSU 85–930 (GenBank accession no. CP003340). No other Rickettsia spp., including R. rickettsii, were detected in any of the 404 ticks tested. The greatest percentage (15%) of R. parkeri–positive A. maculatum ticks were from Fort Campbell. R. parkeri was not detected in any of the A. maculatum ticks from Fort Knox.

Given that A. maculatum ticks were collected at multiple sites during multiple years, and that these ticks have recently been collected in large numbers, this species is probably established in west-central Kentucky and northern Tennessee. To further elucidate its distribution, efforts should be made to collect immature stages of A. maculatum ticks from hosts, particularly birds, throughout both states.

The etiologic agent of Rocky Mountain spotted fever (RMSF), R. rickettsii, was not found in any of the ticks analyzed during this study, a finding that is consistent with findings of Fritzen et al. (11). However, during 2008–2012, a total of 15 human RMSF cases (5-year average rate of 0.1 cases/100,000 population) were reported to the Kentucky Department of Public Health (12). Likewise, for the same period, 1,695 cases of RMSF were reported to the Tennessee Department of Health (5-year average of 393 cases/100,000 population) (13). In addition, an R. parkeri human infection in Kentucky has been confirmed by PCR analysis of a tissue biopsy specimen from a patient (5). Thus, persons in west-central Kentucky and northern Tennessee may be more likely to become infected with a rickettsial agent other than R. rickettsii.

The tick encounter rates during this study suggest that persons entering appropriate habitats, especially for an extended period, are likely to encounter D. variabilis and A. maculatum ticks in west-central Kentucky and northern Tennessee during mid-summer. This study further suggests that although a person is ≈2.5 times more likely to encounter D. variabilis ticks than A. maculatum ticks, persons are ≈4.5 times more likely to encounter an R. parkeri–positive A. maculatum tick than a rickettsia-positive D. variabilis tick. These results are consistent with those of Stromdahl et al. (14).

Further evidence is needed to confirm if R. montanensis in D. variabilis ticks is of medical concern, but there has been 1 report of tick-borne R. montanensis infection associated with a nonfebrile episode in a person with a rash (15). Because of the lack of awareness regarding R. montanensis infection, it is plausible that a rash could be misdiagnosed and assumed to be a sign of a different illness. Even if an illness was recognized as a vectorborne disease, rickettsial serologic assays are not able to distinguish 1 species of SFG rickettsia from another (14). This finding indicates that serologic reactivity caused by exposure to R. montanensis could be attributed to the wrong SFG rickettsiae. Other epidemiologic studies are needed to elucidate how these findings may relate to regional rickettsial illness, but they still confirm that A. maculatum ticks infected with R. parkeri and D. variabilis ticks infected with R. montanensis warrant increased public health awareness in this region.