Prevalence of Ehrlichia ewingii in Amblyomma americanum in North Carolina

Ticks as vectors of Ehrlichia parasites have been the subject of study in North Carolina since 1993. Recently, Ehrlichia ewingii, which causes canine granulocytic ehrlichiosis, was documented as causing human illness in Missouri (3). Previously, the Lone Star tick, Amblyomma americanum, was confirmed as the vector of this parasite in dogs (2). Accordingly, in 1999, we focused our efforts on determining the presence and infection rate of E. ewingii in A. americanum. Field-collected ticks from all three geographical sections of the state were preserved in 95% ethanol. DNA was extracted from 2,970 ticks and subjected to nested PCR analysis (9). DNA from one adult and four pools of nymphs of A. americanum was positive when tested with Ehrlichia groESL primers but failed to amplify with Ehrlichia chaffeensis- or Ehrlichia phagocytophila-like (HGE)-specific primers. The primer set GE2b-GE3x was then used to amplify the 5′ portion of the 16S rRNA gene, giving a characteristic 569-bp product (6). These products were gel purified and sent to Commonwealth Biotechnologies, Inc. (Richmond, Va.), for sequence analysis. The sequences were most closely related to those of E. ewingii when compared to sequences of accession no. {"type":"entrez-nucleotide","attrs":{"text":"U96436","term_id":"2352090"}}U96436 (5) and {"type":"entrez-nucleotide","attrs":{"text":"M73227","term_id":"148300"}}M73227 (1) deposited in GenBank. From one to six base pair differences over a 500+ base pair region were noted between our sequences and the GenBank sequences. After DNA from the five E. ewingii-positive ticks was sequenced, the primer pair EE1-HE3 was used to identify DNA from three additional E. ewingii-positive ticks. A total of eight A. americanum ticks or pools were positive for E. ewingii, while a total of nine were positive for E. chaffeensis. Unlike others (8), we did not find E. ewingii-positive organisms among 1,349 Dermacentor variabilis or 51 Ixodes scapularis ticks tested, even though amplifiable DNA was present. Infection rates for E. ewingii were low in adult A. americanum ticks, 0.4% (1 of 245) in females and 0.9% (2 of 217) in males, and a pool-positive rate of 4.7% (5 of 106) was obtained when 1,308 nymphs in 106 pools were tested for E. ewingii. However, a minimum field infection rate for the nymphs was 0.4% (5 of 1,308 ticks). Positive A. americanum ticks originated in six widely separated piedmont and coastal plain counties (Table ​(Table1),1), showing that E. ewingii has a wide distribution in North Carolina. To date, clinical human infections of E. ewingii have been detected only in Missouri (3), possibly due to the lack of serological diagnostic techniques that differentiate E. chaffeensis from E. ewingii. Considering the high annual incidence rates of human monocytic ehrlichiosis reported for North Carolina (4, 7), the E. ewingii infections in dogs (5), the large populations of A. americanum in North Carolina, and the high levels of human contact with this tick, we suspect that some of those cases of human monocytic ehrlichiosis were caused by E. ewingii. TABLE 1 E. ewingii-positive Lone Star ticks in North Carolina