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Ehrlichiosis and rickettsiosis are two common bacterial tick-borne diseases in the southeastern United States. Ehrlichiosis is caused by ehrlichiae transmitted by Amblyomma americanum and rickettsiosis is caused by rickettsiae transmitted by Amblyomma maculatum and Dermacentor variabilis. These ticks are common and have overlapping distributions in the region. The objective of this study was to identify Anaplasma, Ehrlichia, and Rickettsia species associated with questing ticks in a Rocky Mountain spotted fever (RMSF) hotspot, and identify habitats, time periods, and collection methods for collecting questing-infected ticks. Using vegetation drags and CO 2 -baited traps, ticks were collected six times (May-September 2012) from 100 sites (upland deciduous, bottomland deciduous, grassland, and coniferous habitats) in western Tennessee. Adult collections were screened for Anaplasma and Ehrlichia (simultaneous polymerase chain reaction [PCR]) and Rickettsia using genus-specific PCRs, and resulting positive amplicons were sequenced. Anaplasma and Ehrlichia were only identified within A. americanum (Ehrlichia ewingii, Ehrlichia chaffeensis, Panola Mountain Ehrlichia, and Anaplasma odocoilei sp. nov.); more Ehrlichia-infected A. americanum were collected at the end of June regardless of habitat and collection method. Rickettsia was identified in three tick species; "Candidatus Rickettsia amblyommii" from A. americanum, R. parkeri and R. andeanae from A. maculatum, and R. montanensis ( = montana) from D. variabilis. Overall, significantly more Rickettsia-infected ticks were identified as A. americanum and A. maculatum compared to D. variabilis; more infected-ticks were collected from sites May-July and with dragging. In this study, we report in the Tennessee RMSF hotspot the following: (1) Anaplasma and Ehrlichia are only found in A. americanum, (2) each tick species has its own Rickettsia species, (3) a majority of questing-infected ticks are collected May-July, (4) A. americanum and A. maculatum harbor pathogenic bacteria in western Tennessee, and (5) R. rickettsii remains unidentified.

The population of the lone star tick Amblyomma americanum has expanded in North America over the last several decades. It is known to be an aggressive and nondiscriminatory biter and is by far the most common human-biting tick encountered in Virginia. Few studies of human pathogen prevalence in ticks have been conducted in our state since the mid-twentieth century. We developed a six-plex real-time PCR assay to detect three Ehrlichia species (E. chaffeensis, E. ewingii, and Panola Mountain Ehrlichia) and three spotted fever group Rickettsiae (SFGR; R. amblyommii, R. parkeri, and R. rickettsii) and used it to test A. americanum from around the state. Our studies revealed a presence of all three Ehrlichia species (0-24.5%) and a high prevalence (50-80%) of R. amblyommii, a presumptively nonpathogenic SFGR, in all regions surveyed. R. parkeri, previously only detected in Virginia's Amblyomma maculatum ticks, was found in A. americanum in several surveyed areas within two regions having established A. maculatum populations. R. rickettsii was not found in any sample tested. Our study provides the first state-wide screening of A. americanum ticks in recent history and indicates that human exposure to R. amblyommii and to Ehrlichiae may be common. The high prevalence of R. amblyommii, serological cross-reactivity of all SFGR members, and the apparent rarity of R. rickettsii in human biting ticks across the eastern United States suggest that clinical cases of tick-borne disease, including ehrlichiosis, may be commonly misdiagnosed as Rocky Mountain spotted fever, and that suspicion of other SFGR as well as Ehrlichia should be increased. These data may be of relevance to other regions where A. americanum is prevalent.