title: Prevalence of Rickettsiales in ticks removed from the skin of outdoor workers in North Carolina.
abstract: 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.
authors.affiliation: Department of Entomology, North Carolina State University, Campus Box 7647, Raleigh, NC, 27695-7647, USA. slee19@ncsu.edu.|Present address: National Institutes of Health, Bethesda, MD, 20892, USA. slee19@ncsu.edu.
authors.forename: Sangmi
authors.initials: S
authors.lastname: Lee
authors.affiliation: Department of Entomology, North Carolina State University, Campus Box 7647, Raleigh, NC, 27695-7647, USA. mlkakuma@ncsu.edu.
authors.forename: Madhavi L
authors.initials: ML
authors.lastname: Kakumanu
authors.affiliation: Department of Entomology, North Carolina State University, Campus Box 7647, Raleigh, NC, 27695-7647, USA. loganathan_ponnusamy@ncsu.edu.
authors.forename: Loganathan
authors.initials: L
authors.lastname: Ponnusamy
authors.affiliation: Department of Epidemiology, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, NC, 27599, USA. vaughn.meagan@gmail.com.
authors.forename: Meagan
authors.initials: M
authors.lastname: Vaughn
authors.affiliation: Department of Epidemiology, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, NC, 27599, USA. sheana@nc.rr.com.
authors.forename: Sheana
authors.initials: S
authors.lastname: Funkhouser
authors.affiliation: Department of Entomology, North Carolina State University, Campus Box 7647, Raleigh, NC, 27695-7647, USA. hethornt@ncsu.edu.
authors.forename: Haley
authors.initials: H
authors.lastname: Thornton
authors.affiliation: Department of Epidemiology, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, NC, 27599, USA. meshnick@email.unc.edu.
authors.forename: Steven R
authors.initials: SR
authors.lastname: Meshnick
authors.affiliation: Department of Entomology, North Carolina State University, Campus Box 7647, Raleigh, NC, 27695-7647, USA. apperson@ncsu.edu.
authors.forename: Charles S
authors.initials: CS
authors.lastname: Apperson
mesh_terms: D000818:Animals; D001096:Arachnid Vectors / Q000382:microbiology*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D004311:Double-Blind Method; D005260:Female; D006801:Humans; D026863:Ixodidae / Q000382:microbiology*; D008297:Male; D008969:Molecular Sequence Data; D009657:North Carolina / Q000453:epidemiology; D016133:Polymerase Chain Reaction; D015995:Prevalence; D012281:Rickettsia / Q000235:genetics / Q000302:isolation & purification*; D012282:Rickettsia Infections / Q000453:epidemiology* / Q000382:microbiology; D017422:Sequence Analysis, DNA; D013984:Tick Infestations / Q000453:epidemiology*
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal