[Emerging Infectious Diseases * Volume 4 * Number 1 * January - March 1998] Dispatches Prevalence of Tick-Borne Pathogens in _Ixodes scapularis_ in a Rural New Jersey County Shobha Varde,* John Beckley,† and Ira Schwartz* *New York Medical College, Valhalla, New York, USA; and †Department of Health, County of Hunterdon, Flemington, New Jersey, USA --------------------------------------------------------------------------- To assess the potential risk for other tick-borne diseases, we collected 100 adult _Ixodes scapularis_ in Hunterdon County, a rapidly developing rural county in Lyme disease–endemic western New Jersey. We tested the ticks by polymerase chain reaction for _Borrelia burgdorferi_, _Babesia microti_, and the rickettsial agent of human granulocytic ehrlichiosis (HGE). Fifty-five ticks were infected with at least one of the three pathogens: 43 with _B. burgdorferi_, five with _B. microti_, and 17 with the HGE agent. Ten ticks were coinfected with two of the pathogens. The results suggest that county residents are at considerable risk for infection by a tick-borne pathogen after an _I. scapularis_ bite. The vector-borne diseases Lyme disease, human babesiosis, and human granulocytic ehrlichiosis (HGE) are emerging in the Northeast and upper Midwest regions of the United States (1,2). The etiologic agents for these diseases (_Borrelia burgdorferi_, _Babesia microti_, and the HGE agent, respectively) appear to share the same vertebrate reservoir host (_Peromyscus leucopus_) and tick vector (_Ixodes scapularis_) (3-6). Immunologic evidence of human coinfection with these pathogens has been reported (7-10), and a culture-confirmed case of coinfection with _B. burgdorferi_ and the HGE agent has recently been described (11). Thus, in Lyme disease–endemic areas, there may be a substantial risk for coinfection with _B. burgdorferi_ and either _B. microti_ or the HGE-causing rickettsia. Hunterdon County (population 118,000) is a rural, but rapidly developing, county in western New Jersey (Figure), with many homes in wooded settings. In 1996, the county had the third highest case rate of Lyme disease (524 per 100,000) of all counties in the United States (Centers for Disease Control and Prevention case reports). The county has also had many suspected, but no serologically confirmed, cases of HGE. [fig] Figure. Map of New Jersey showing Hunterdon County. Black dots indicate tick collection sites. The risk of acquiring a tick-borne pathogen in a specific geographic location depends largely on tick density and the prevalence rate of the agent in the tick population. Although cultivation of a microbial agent is considered optimal, molecular detection is more practical since it permits the rapid assay of large numbers of individual specimens. We report here the prevalence rate of _B. burgdorferi_, _B. microti_, and the agent of HGE in _I. scapularis_ in Hunterdon County by species-specific polymerase chain reaction (PCR). One hundred adult _I. scapularis_ were collected by drag cloth or from personal clothing at 10 sites in the county during the fall of 1996 (Figure). Ticks were stored at room temperature in 70% ethanol until analysis. DNA was isolated from individual ticks with the Isoquick DNA extraction kit (ORCA Research, Bothell, WA) (12). The final DNA pellets were resuspended in 50 µl of sterile water, and a 10-µl aliquot was used for each PCR test. _B. burgdorferi_–specific PCR used primers IS1 and IS2 (13). The HGE agent was detected by PCR using primers GER3 and GER4, as described by Munderloh et al. (14), and _B. microti_ DNA was detected according to methods used by Persing et al. (15). PCR products were electrophoresed on 2% agarose gels stained with ethidium bromide. For _B. burgdorferi_ and _B. microti_, hybridization with specific probes was carried out after transfer to nylon membranes (13). In each PCR experiment appropriate negative controls were used; these included master mix controls lacking template and reactions containing _B. burgdorferi_ DNA, HGE agent DNA, or _Escherichia coli_ DNA as additional controls for HGE agent, _B. burgdorferi_, or _B. microti_ PCR, respectively. To prevent cross-contamination, specimen preparation, PCR amplification, and post-PCR analysis were performed in separate laboratories. Results of PCR analysis for each of the pathogens on individual ticks are presented in the Table. At least one of the three pathogens was present in 55% of the ticks. The highest prevalence rate was that for _B. burgdorferi_ (43/100), followed by the agent of HGE (17/100) and _B. microti_ (5/100). Furthermore, these pathogens were widely distributed. At least one tick at each site was infected with _B. burgdorferi_, nine of 10 sites had HGE agent–positive ticks, and _B. microti_–positive ticks were found at five different collection sites. Ten ticks were coinfected with two of the three agents; no ticks were infected with all three pathogens. The _B. microti_ PCR amplification products were gel purified and subjected to automated DNA sequencing. The 238 bp products yielded sequences identical to that reported for _B. microti_ 18S ribosomal DNA (15). Table. Prevalence rate of _Borrelia burgdorferi_, human granulocytic ehrlichiosis (HGE) agent, and _Babesia microti_ in 100 adult _Ixodes scapularis_ ------------------------------------------------------------------ Pathogen No. infected ticks ------------------------------------------------------------------ _B. burgdorferi_ 43 HGE agent 17 _B. microti_ 5 _B. burgdorferi_ and HGE agent 6 _B. burgdorferi_ and _B. microti_ 2 HGE agent and _B. microti_ 2 _B. burgdorferi_, HGE agent, and _B. microti_ 0 ------------------------------------------------------------------ The prevalence rates for the three pathogens reported here are consistent with earlier studies at selected sites in the northeastern United States. Telford et al. reported prevalence rates of 36%, 11%, and 9% for _B. burgdorferi_, the HGE agent, and _B. microti_, respectively, in adult _I. scapularis_ on Nantucket Island, Massachusetts; coprevalence of _B. burgdorferi_ and the HGE agent was 4%, and no simultaneous infection with _B. burgdorferi_ and _B. microti_ was observed (4). We have reported prevalence rates of 52% and 53% for _B. burgdorferi_ and the HGE pathogen, and coprevalence of 26% at a site in Westchester County, New York (5). Given the simultaneous infection of _I. scapularis_ with these pathogens, it is not surprising that numerous serosurveys of Lyme disease patients indicate the coincident presence of antibodies to _B. burgdorferi_ and _B. microti_ and human granulocytic ehrlichia (7-10). These findings do not establish concurrent, active infection, nor do they demonstrate simultaneous transmission by a single tick bite; however, they suggest that persons living in disease-endemic areas are exposed to these agents, presumably as a result of tick bites. Human infection by any of the three tick-borne agents alone generally results in similar acute manifestations, including fever, headache, and myalgia (1,10,16). Reported cases of Lyme disease greatly outnumber those of babesiosis or HGE. This, in conjunction with seroepidemiologic data, suggests that many infections with _B. microti_ and granulocytic ehrlichia are subclinical. Dual infection with _B. burgdorferi_ and _B. microti_ often results in more severe illness (10); whether this is also true for concurrent infection by the agents of Lyme disease and HGE remains to be established. Human coinfection by multiple tick-borne agents may account for the variable nature of the clinical manifestations of Lyme disease. This study demonstrates that the agents of Lyme disease, human babesiosis, and HGE coexist in the tick population of a previously identified Lyme disease–endemic region of New Jersey. The study provides further evidence that these three pathogens may be prevalent throughout the range of _I. scapularis._ Infection with any of these three tick-borne pathogens should be considered for residents or visitors of a disease-endemic area who have flulike symptoms and a history of a tick bite. Acknowledgments We thank Gary Wormser and Dennis White for helpful comments, Chris Kolbert and David Persing for providing advice and positive controls for _B. microti_ PCR, and the Hunterdon County Tickborne Disease Research Group and Hunterdon Medical Center for their support. This work was supported in part by NIH grant AR41511. References 1. Walker DH, Barbour AG, Oliver JH, Lane RS, Dumler JS, Dennis DT, et al. Emerging bacterial zoonotic and vector-borne diseases: ecological and epidemiological factors. JAMA 1996;275:463-9. 2. Persing DH, Conrad PA. Babesiosis: new insights from phylogenetic analysis. Infect Agents Dis 1995;4:182-95. 3. Anderson J, Mintz E, Gadbaw J, Magnarelli L._ Babesia microti_, human babesiosis and _B. burgdorferi_ in Connecticut. J Clin Microbiol 1991;29:2779-83. 4. Telford SR III, Dawson JE, Katavolos P, Warner CK, Kolbert CP, Persing DH. Perpetuation of the agent of human granulocytic ehrlichiosis in a deer tick-rodent cycle. Proc Natl Acad Sci U S A 1996;93:6209-14. 5. Schwartz I, Fish D, Daniels TJ. Prevalence of the rickettsial agent of human granulocytic ehrlichiosis in ticks from a hyperendemic focus of Lyme disease [letter]. N Engl J Med 1997;337:49-50. 6. Piesman J, Mather TN, Telford SR III, Spielman A. Concurrent _Borrelia burgdorferi_ and _Babesia microti_ infection in nymphal _Ixodes dammini_. J Clin Microbiol 1986;24:446-7. 7. Benach JL, Coleman JL, Habicht GS, MacDonald A, Grunwaldt E, Giron JA. Serological evidence for simultaneous occurrences of Lyme disease and babesiosis. J Infect Dis 1985;152:473-7. 8. Magnarelli LA, Dumler JS, Anderson JF, Johnson RC, Fikrig E. Coexistence of antibodies to tick-borne pathogens of babesiosis, ehrlichiosis, and Lyme borreliosis in human sera. J Clin Microbiol 1995;33:3054-7. 9. Mitchell PD, Reed KD, Hofkes JM. Immunoserologic evidence of coinfection with _Borrelia burgdorferi, Babesia microti_, and human granulocytic _Ehrlichia_ species in residents of Wisconsin and Minnesota. J Clin Microbiol 1996;34:724-7. 10. Krause PJ, Telford SR III, Spielman A, Sikand V, Ryan R, Christianson D, et al. Concurrent Lyme disease and babesiosis. Evidence for increased severity and duration of illness. JAMA 1996;275:1657-60. 11. Nadelman RB, Horowitz HW, Hsieh TC, Wu JM, Aguero-Rosenfeld ME, Schwartz I, et al. Simultaneous human granulocytic ehrlichiosis and Lyme borreliosis. N Engl J Med 1997;337:27-30. 12. Schwartz I, Varde S, Nadelman RB, Wormser GP, Fish D. Inhibition of efficient polymerase chain reaction amplification of _Borrelia burgdorferi_ DNA in blood-fed ticks. Am J Trop Med Hyg 1997;56:339-42. 13. Schwartz I, Wormser GP, Schwartz JJ, Cooper D, Weissensee P, Gazumyan A, et al. Diagnosis of early Lyme disease by polymerase chain reaction amplification and culture of skin biopsies from erythema migrans lesions. J Clin Microbiol 1992;30:3082-8. 14. Munderloh UG, Madigan JE, Dumler JS, Goodman JL, Hayes SF, Barlough JE, et al. Isolation of the equine granulocytic ehrlichiosis agent, _Ehrlichia equi_, in tick cell culture. J Clin Microbiol 1996;34:664-70. 15. Persing D, Mathiesen D, Marshall W, Telford S, Spielman A, Thomford J, Conrad P. Detection of _Babesia microti_ by polymerase chain reaction. J Clin Microbiol 1992;30:2097-103. 16. Boustani MR, Gelfand JA. Babesiosis. Clin Infect Dis 1996;22:611-5. --------------------------------------------------------------------------- Emerging Infectious Diseases National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, GA URL: ftp://ftp.cdc.gov/pub/EID/vol4no1/ascii/varde.txt Please note that figures and equations are not available in ASCII format; their placement within the text is noted by [fig] and [eq], respectively. Greek symbols are spelled out. The following codes are used: (ft) for footnote; (sup) for superscript; (sub) for subscript; >/= for greater than or equal to. Italics are indicated by underlining before and after the italicized word(s) (e.g., _E.coli_ for E. coli).