Results
Fifty (13.3%) of 375 I. scapularis ticks from Bridgeport, Conn., were PCR positive for ehrlichiae ( Table 1 ). The percentage positive in each of the 4 years ranged from 6.1% in nymphs in 1998 to 23.3% in adults in 1996. Less year-to-year variation was noted in adult ticks, in which infection prevalence ranged from 11.7% (1997) to 23.3% (1996). PCR analysis of EDTA blood samples from white-footed mice collected in Connecticut during summer and fall 1997 and spring 1998 showed that 17 (36.2%) of 47 in 1997 and 3 (60%) of 5 in 1998 were positive[22]. The amplification products were sequenced for each Ehrlichia PCR-positive mouse and tick. All products from samples collected in the Bridgeport, Conn., area from 1996 through 1998 had sequences identical to the 16S rRNA gene (EP-ha) previously amplified and sequenced from documented human infections in the Northeast and Upper Midwest United States and in Europe[4]. The 16S rRNA sequence determined from adult ticks collected from Bridgeport in 1999 showed that all 12 positive samples also contained the human agent (EP-ha), although one of the ticks produced a mixed sequence, suggesting the presence of more than one agent. The PCR products from this tick were cloned, and individual clones were purified and sequenced. These data confirmed the presence of a mixed population of ehrlichiae containing some 16S rRNA sequences that matched EP-ha and some that differed from EP-ha by two nucleotides. The latter sequence was identical to a variant (called variant 1) previously described in ticks in Rhode Island and deer in Maryland and Wisconsin[13,14] ( Table 2 ). In contrast to ticks and rodents from the Bridgeport area, nymphal ticks collected in 1997 from Bluff Point in southeastern Connecticut contained a nearly equal distribution of EP-ha (5 [55.6%] of 9 positives) and variant 1 (4 [44.4%] of 9 positives) ehrlichiae.
Rhode Island samples from I. scapularis ticks, white-footed mice, and chipmunks contained E. phagocytophila variants as well as EP-ha. A total of 123 (22.9%) of 538 ticks were positive for E. phagocytophila by PCR, including 61 (26.3%) of 232 adults and 62 (20.3%) of 306 nymphs. DNA sequencing was performed on 92 of these PCR products, and overall, only 24 (26.1%) showed sequences identical to those of EP-ha. Fifteen (16.3%) ticks showed sequences corresponding to variant 1. The rest of the ticks (53 [57.6%]) had a novel sequence differing from EP-ha by 2 nucleotides and from variant 1 by 4 nucleotides (hereafter called variant 2) ( Table 2 ).
PCR testing of blood samples from 19 Rhode Island chipmunks in 1996 detected 11 (57.9%) positives. DNA sequencing of these PCR products showed that nine were identical to the sequence of EP-ha; the remaining two represented novel variant sequences, each differing from EP-ha by a single nucleotide (variants 3 and 4; Table 2 ). Although both the white-tailed deer agent and the E. equi/CA human sequence variant ( Table 2 ) are amplified by the PCR assay used in this study, neither agent has been detected in potential rodent reservoir populations in Connecticut or Rhode Island. Host and vector associations of EP-ha and the four E. phagocytophila variants found in Rhode Island are shown in Table 3 .
The prevalence of E. phagocytophila in I. scapularis ticks (adults and nymphs combined, years 1996-1999) was higher in Rhode Island (22.8%) than in Bridgeport (13.3%) (p<0.001; Fisher's exact test). This finding was also true for adult ticks: 26.3% were infected in Rhode Island compared with 15.4% in Bridgeport (p=0.002). Using either the total number of ticks tested or adult ticks only, the prevalence of E. phagocytophila in Rhode Island compared with Bridgeport was 1.7. However, if the 1997 Rhode Island data, which were skewed by an unusually large number of variants, are removed from the calculations, the percentage of E. phagocytophila-positive ticks (adults and nymphs) was significantly higher in Bridgeport (13.3%) than in Rhode Island (8.2%) (p=0.03). The same analysis, when restricted to the adult tick population, showed no significant difference between Connecticut (15.4%) and Rhode Island (13.4%) EP-positive ticks (p=0.6). If the 1997 Rhode Island data are excluded, the prevalence of E. phagocytophila in that state compared with Bridgeport was 0.6 for the total number of ticks tested and 0.8 for adult ticks only.
Infection prevalence data were available for adult and nymphal ticks from the same site for 3 years: Rhode Island in 1997 and 1999 and Bridgeport in 1998 ( Table 1 ). For two of these, Rhode Island in 1999 and Bridgeport in 1998, the prevalence of E. phagocytophila was significantly higher (Rhode Island 1999; p=0.01) or borderline higher (Connecticut 1998; p=0.065) in adults than in nymphs. E. phagocytophila infection rates in nymphal and adult ticks from Rhode Island in 1997 did not differ significantly (p=0.21).
Temporal trends in tick infection rates showed that the prevalence of E. phagocytophila in Rhode Island nymphs was highest in 1997 and then declined in 1998 and 1999 (p<0.001; chi-square test for trend). However, the high number of variants found in both adult and nymphal ticks from Rhode Island in 1997 influenced this analysis, as E. phagocytophila prevalence in Rhode Island in 1997 was significantly higher than in all other years combined (p<0.001). In Bridgeport, no significant temporal trends were noted in E. phagocytophila infection rates in adult ticks (p=0.3), nor were significant prevalence or temporal trends noted in the rodents tested from any of the sites.
Analysis of the proportion of E. phagocytophila-positives that were variants showed that prevalence of the variants in Rhode Island (73.9% variants) was significantly higher than in Bridgeport (0.02% variants) (p<0.001). When the proportion of E. phagocytophila-positives that were variants was compared with the total number of positives for the two Connecticut sites, the ticks from Bluff Point (44.4% variants) showed significantly higher rates than ticks from Bridgeport (0.02% variants) (p<0.001).
Emerging Infectious Diseases. 2002;8(5) © 2002 Centers for Disease Control and Prevention (CDC)
Cite this: Genetic Variants of Ehrlichia phagocytophila, Rhode Island and Connecticut - Medscape - May 01, 2002.
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