Genetic Variants of Ehrlichia phagocytophila, Rhode Island and Connecticut

Discussion

Strains of E. phagocytophila found in nature are capable of causing disease in sheep, cattle, horses, dogs, cats, and humans. These strains, including the species previously known as the HGE agent and E. equi, are grouped as a single species on the basis of their close relationship at the genetic and antigenic levels. However, biological and ecological differences clearly exist between strains of E. phagocytophila, including varying host pathogenicity, vectors, DNA sequence, and geographic distribution. Small ribosomal subunit (16S) DNA sequences are highly conserved in bacteria and are often used to identify and differentiate bacterial species. The 16S rRNA gene sequences amplified from every confirmed human case, except for two isolated cases in northern California, have been identical to the E. phagocytophila-human agent (EP-ha) sequence determined by Chen et al.^[4]. PCR-positive white-footed mice (n=20) and I. scapularis ticks (n=38) collected in Bridgeport from 1996 through 1998 also harbored only E. phagocytophila identical in sequence to EP-ha for a 546-bp region of the 16S rRNA gene^[4]. Sequence analysis of PCR products from two Connecticut deer blood samples showed DNA identical to the EP-ha p44 gene sequence^[23]. However, an Ehrlichia organism with a 16S rRNA gene sequence differing from EP-ha by a single nucleotide has been identified in white-tailed deer from Maryland and Wisconsin and in I. scapularis from Rhode Island^[13,14].

In contrast to our results from Bridgeport, where we consistently found EP-ha, mice and ticks from Rhode Island had a significantly lower percentage of isolates identical to EP-ha, but several E. phagocytophila variants with novel 16S rRNA gene sequences. These data indicate that variant forms of E. phagocytophila, not yet associated with human or veterinary disease, frequently occur in Rhode Island. The same or additional E. phagocytophila variants may also occur in other regions of the United States, but this concept remains to be investigated.

Most PCR assays amplify products from the variant agents that are the same size as the EP-ha PCR product, so that variants are indistinguishable when the products are analyzed only by agarose gel electrophoresis. Therefore, results from other human-infection prevalence surveillance studies in ticks and rodents that have not included PCR product sequencing may be misleading. For example, if we had not sequenced our PCR products for 1997, we would have concluded that 46.4% of nymphal and 38.3% of adult I. scapularis ticks collected in southern Rhode Island were positive for EP-ha. Actually, only 11.9% of the positives that were sequenced and an estimated 5.0% of the total ticks tested were EP-ha positive, with the rest of the 1997 Rhode Island positives consisting of genetic variants not yet associated with human disease.

The 16S rRNA sequences obtained from tick and rodent samples collected from Bridgeport from 1996 through 1998 were identical to EP-ha. However, in 1999, one tick collected in that site was positive for both EP-ha and a variant (variant 1) previously found in Rhode Island. In a retrospective analysis of ticks collected at another eastern Connecticut site (Bluff Point) close to the Rhode Island border, variant 1 was found in 1997. Our inability to detect variant 1 despite extensive testing of samples collected in Bridgeport from 1996 to 1998 and its subsequent appearance in 1999 suggests that its geographic range may be expanding westward. Additional studies with larger sample sizes of ticks and rodents from Bridgeport and other locations in Connecticut are needed to assess the prevalence of EP-ha and the variants, as our Bridgeport results may not be representative of the entire state. In fact, the Bluff Point data suggest that other areas of Connecticut may have EP-ha/variant populations quite different from those in Bridgeport and more similar to the distribution noted in Rhode Island.

The identification of the coinfected tick in Connecticut represents the first detection of more than one strain of E. phagocytophila in a single tick vector in the United States, although the coinfection of Ixodes ricinus ticks by 2 E. phagocytophila strains has been reported in Europe^[24]. These data indicate that two strains of the agent are capable of coexisting in a single tick, at least transiently, and that they can survive the molting process, since the coinfection was found in an unfed, host-seeking adult tick.

The results from Rhode Island samples collected in 1997 are unusual in several regards. First, the rate of E. phagocytophila-positive ticks (42.2% nymphs and adults) was very high relative to all other tick populations sampled from 1996 through 1999, and many of the positives were variant 2 (79.1% of PCR-positives sequenced). Second, the 1997 Rhode Island ticks represent the only population in which E. phagocytophila prevalence was higher in nymphs than adults (46.4% nymphs and 38.3% adults). Finally, variant 2 sequences were also seen in samples collected in 1997 from white-footed mice and chipmunks but were not detected before and have not been detected after 1997. The fact that both nymphal and adult questing ticks were positive for variant 2 suggests that the variant was present in reservoir species during both larval and nymphal feedings and may have been present in reservoirs from late summer 1996 through summer 1997. Why this variant appeared only in Rhode Island during 1997, was the most prevalent strain infecting ticks that year, and then completely disappeared are matters of speculation. Variant 2 may be a more common infection in a reservoir species that we did not examine, which may be less commonly targeted by host-seeking ticks. Expression of variant 2 in the tick population could have resulted if, during 1996-1997, host populations preferred by immature I. scapularis (i.e., white-footed mice and chipmunks) were lower than normal, resulting in a higher proportion of ticks feeding on such atypical hosts harboring variant 2. After molting, nymphs infected as larvae the previous year could have transmitted variant 2 to the more preferred hosts of immature ticks, resulting in the variant 2-positive mice found in 1997. Subsequent reestablishment of normal host populations may then have diluted the prevalence of variant 2, as immature tick feeding reverted to the preferred hosts.

Although the function and biological importance of the genetic differences in E. phagocytophila strains are unknown, we hypothesize that the variants may be interfering with maintenance and transmission of the human disease-causing agent (EP-ha). Even if an increased human ehrlichiosis case surveillance effort in Connecticut is taken into account, the number of confirmed and suspected cases differs dramatically between the two neighboring states: several hundred cases were reported in Connecticut compared with fewer than 25 cases in Rhode Island during the same time period. From 1995 to 1997, 178 cases were confirmed or suspected^[25], and case reports in Connecticut increased substantially in 1998 (228 provisional, 104 confirmed, Connecticut Dept. of Public Health). These adjacent states share many of the ecologic factors that support natural maintenance of both Borrelia burgdorferi and granulocytic ehrlichiae, such as populations of the tick vector I. scapularis and reservoir rodents, including white-footed mice (P. leucopus)^[19,22]. The incidence of Lyme disease in Connecticut and Rhode Island has been the highest in the nation for several years, with Connecticut having a reported incidence only approximately 1.3-1.5 times higher than Rhode Island's (26). In contrast, through 1997 there was a 24-fold difference in the incidence of reported HGE cases in the two states (Connecticut 15.9; Rhode Island 0.67). Therefore, the E. phagocytophila variants may have a competitive advantage over the EP-ha, possibly in infecting certain reservoir or vector populations. A lower incidence of EP-ha and less human disease would therefore be expected in areas where the variants predominate, since a lower proportion of ticks would harbor EP-ha.

Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever, was first identified in the early 1900s on the basis of its association with human disease^[27]. Subsequent studies of veterinary infections and tick populations identified numerous Rickettsia species closely related to R. rickettsii, all clearly members of the spotted fever group but not associated with human disease. These species include R. montana, R. rhiphicephali, R. parkeri, R. bellii, and the "east side agent" R. peacockii^[28,29]. Nonpathogenic rickettsiae are thought to interfere with the development of more virulent R. rickettsii in Dermacentor ticks and may be found more often in ticks than are the more virulent species^[30,31]. Our data suggest that a similar situation may exist among the granulocytic ehrlichiae, with both pathogenic and nonpathogenic genetic variants coexisting in nature. Isolation of the new variants will allow us to address this competitive-advantage hypothesis experimentally in both ticks and mice through the use of mixed infections in the laboratory.

Identification and use of novel gene targets more variable than the 16S rRNA gene will eventually permit better assessment of variability between strains of E. phagocytophila ^[32,33,34]. Future studies should include E. phagocytophila from additional geographic areas where a substantial number of human cases of granulocytic ehrlichiosis are reported (e.g., New York, Wisconsin, Minnesota) compared with areas (e.g., New Jersey, Pennsylvania, Delaware, Maryland, California) with similar vector densities but with little or no human disease.

¹ Since this study was conducted, new nomenclature ( Anaplasma phagocytophila) has been proposed; see Dumler JS, Barbet AF, Bekker CP, Dasch GA, Palmer GH, Ray SC, et al. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia, and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and 'HGE agent' as subjective synonyms of Ehrlichia pagocytophila. Int J Syst Evol Microbiol 2001;51:2145-65.

The authors thank Stacey Carlton, Nathan Miller, Chris Whitehouse, Susan Hiers, Katya Mason, Meaghan McKenna, Kim Bizzell, Jennifer Hatfield, Rachael Priestly, Michael Brewer, and Neeta Pardanani for technical assistance. We are grateful to the Biotechnology Core Facility of the National Center for Infectious Diseases, CDC, for the synthesis of oligonucleotides.

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.