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Background  Tick-transmitted rickettsial diseases, such as ehrlichiosis and spotted fever rickettsiosis, are significant sources of morbidity and mortality in the southern United States. Because of their exposure in tick-infested woodlands, outdoor workers experience an increased risk of infection with tick-borne pathogens. As part of a double blind randomized-controlled field trial of the effectiveness of permethrin-treated clothing in preventing tick bites, we identified tick species removed from the skin of outdoor workers in North Carolina and tested the ticks for  Rickettsiales  pathogens.    Methods  Ticks submitted by study participants from April-September 2011 and 2012 were identified to species and life stage, and preliminarily screened for the genus  Rickettsia  by nested PCR targeting the 17-kDa protein gene.  Rickettsia  were further identified to species by PCR amplification of 23S-5S intergenic spacer (IGS) fragments combined with reverse line blot hybridization with species-specific probes and through cloning and nucleotide sequence analysis of 23S-5S amplicons. Ticks were examined for  Ehrlichia  and  Anaplasma  by nested PCR directed at the  gltA,  antigen-expressing gene containing a variable number of tandem repeats, 16S rRNA, and  groESL  genes.    Results  The lone star tick ( Amblyomma americanum ) accounted for 95.0 and 92.9% of ticks submitted in 2011 ( n  = 423) and 2012 ( n  = 451), respectively. Specimens of American dog tick ( Dermacentor variabilis ), Gulf Coast tick ( Amblyomma maculatum ) and black-legged tick ( Ixodes scapularis ) were also identified. In both years of our study, 60.9% of ticks tested positive for 17-kDa. “ Candidatus  Rickettsia amblyommii”, identified in all four tick species, accounted for 90.2% (416/461) of the 23S-5S-positive samples and 52.9% (416/787) of all samples tested. Nucleotide sequence analysis of  Rickettsia -specific 23S-5S IGS,  ompA  and  gltA  gene fragments indicated that ticks, principally  A. americanum , contained novel species of  Rickettsia . Other  Rickettsiales , including  Ehrlichia ewingii ,  E. chaffeensis ,  Ehrlichia  sp. (Panola Mountain), and  Anaplasma phagocytophilum , were infrequently identified, principally in  A. americanum .    Conclusions  We conclude that in North Carolina, the most common rickettsial exposure is to  R. amblyommii  carried by  A. americanum . Other  Rickettsiales  bacteria, including novel species of  Rickettsia , were less frequently detected in  A. americanum  but are relevant to public health nevertheless.    Electronic supplementary material  The online version of this article (doi:10.1186/s13071-014-0607-2) contains supplementary material, which is available to authorized users.

Prevalence of Rickettsiales in ticks removed from the skin of outdoor workers in North Carolina