Babesia microti, Human Babesiosis, and Borrelia burgdorferi

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1991, p /91/ $02.00/0 Copyright C 1991, American Society for Microbiology Vol. 29, No. 12 Babesia microti, Human Babesiosis, and Borrelia burgdorferi in Connecticut JOHN F. ANDERSON,'* ERIC D. MINTZ,2 JOSEPH J. GADBAW,3 AND LOUIS A. MAGNARELLI' Department of Entomology, The Connecticut Agricultural Experiment Station, New Haven, Connecticut ; Epidemiology Section, Connecticut Department of Health Services, Hartford, Connecticut 06106, and Division of Field Epidemiology, Epidemiology Program Office, Centers for Disease Control, Atlanta, Georgia ; and Lawrence and Memorial Hospital, New London, Connecticut Received 10 June 1991/Accepted 13 September 1991 Babesia microti was isolated from a white-footed mouse (Peromyscus leucopus) that was captured in southeastern Connecticut in 1988, when the first human case of babesiosis acquired in Connecticut was recognized. To date, 13 cases of babesiosis have been reported in Connecticut, the largest number of human cases reported on the mainland United States. Two of nine patients queried remembered a prior tick bite. Since Babesia parasites are known to be vectored only by ticks, we surmise that 12 of these infections were acquired via tick bites; 1 was obtained by blood transfusion (the patient was 46 years of age) from an endemically infected donor. The ages of the patients with tick-acquired babesiosis ranged from 61 to 95 years. Two patients died with active infections, and one patient died from chronic obstructive pulmonary disease soon after treatment with clindamycin and quinine. Indirect fluorescent-antibody titers of blood samples drawn at the time of hospitalization for 11 patients and at the time of active infection for 1 asymptomatic person ranged from 1:1,024 to 1:4,096. Five of eight patients with babesiosis also had significant immunoglobulin G or immunoglobulin M titers (1:640 to 1:5,120) to Borrelia burgdorferi. B. microti was isolated in Syrian hamsters inoculated with blood from 7 of 12 patients tested and was also isolated from mice captured in six towns. The peridomestic nature of the disease was demonstrated by isolating the parasite from white-footed mice captured in or near the yards of eight different patients. Of 59 mice tested, 27 were positive and 25 were coinfected with B. burgdorferi. The isolation of B. microti from a white-footed mouse captured in north-central Connecticut (West Hartford), away from the focus of human infections in southeastern Connecticut, suggests that this pathogen may spread into other areas where Ixodes dammini, the tick vector, becomes established. Two new tick-associated illnesses were reported from widely different geographic regions of the United States in Erythema chronicum migrans, now known as Lyme disease (32), was reported in Wisconsin (26) and human babesiosis was documented on Nantucket Island, Mass. (35). Lyme disease is a multisystemic illness affecting joint, heart, skin, nervous system, and other tissues (30), while babesiosis is a malarialike illness that often results in headache, malaise, anemia, and fatigue (35). The two etiologic agents, Borrelia burgdorferi (11, 18) and Babesia microti (35), were subsequently demonstrated to be transmitted by the same species of tick, Ixodes dammini (11, 28, 31). While B. burgdorferi has become the most common vector-borne pathogen in the United States, with 4,572 cases being reported from 43 states in 1988 (21), less than 250 cases of babesiosis caused by B. microti are known (16). Almost all of the tick-associated cases of babesiosis have been acquired on islands in the northeastern United States. Exceptions include two cases reported in Wisconsin (33) and two cases documented on Cape Cod, Mass. (13). In 1988, a possible new endemic focus of B. microti on the mainland of the United States was recognized when a single clinical case of babesiosis was reported in a Stonington, Conn., resident with no recent travel history. In the summer of 1989, four additional cases were reported in residents of the same town, and a fifth case was reported in a resident of Old Lyme, Conn. (15). By December 1989, a total of nine cases had been reported in Connecticut residents. Addition- * Corresponding author ally, seroprevalence studies of persons with positive anti-b. burgdorferi titers suggested a high potential for exposure to B. microti. In one study (19), 9.5% of persons tested statewide had elevated antibody titers (21:64) to B. microti. In another study (23), 37% of persons tested who lived in the same towns as patients with clinical babesiosis had elevated titers (21:64) to B. microti. We now report (i) the largest number of babesiosis cases to date that have been contracted on the mainland United States, (ii) the isolation of B. microti from humans living in southeastern Connecticut, (iii) the isolation of B. microti from white-footed mice (Peromyscus leucopus) captured in or near the yards of humans diagnosed with babesiosis in southeastern Connecticut and from woodlands in southeastern and north-central Connecticut, (iv) the isolation of B. microti from a mouse captured in Connecticut as early as 1988, and (v) a relatively high prevalence of white-footed mice dually infected with B. microti and B. burgdorferi. MATERIALS AND METHODS Attempted isolation of B. microti from humans. Blood drawn from 11 patients suspected of infection with B. microti was placed into EDTA tubes, and blood drawn from one patient with babesiosis was placed into a tube without anticoagulant. Blood smears from 12 patients were stained with Wright's stain and examined microscopically for Babesia parasites. The time that blood was drawn for attempted isolation of Babesia parasites relative to the time of illness in the 12 patients was variable, ranging from the first day of hospitalization to almost 1 year after the onset of disease. A

2 2780 ANDERSON ET AL. total of 0.5 to 1 ml of uncoagulated blood from each person was inoculated intraperitoneally into one 3- to 6-week-old Syrian hamster for attempted isolation of B. microti (10). The clotted blood sample was shaken, and clumps of erythrocytes were drawn into a syringe. A total of 0.2 ml of clotted blood and serum was inoculated intraperitoneally into one hamster. Blood smears were obtained from tail snips from each inoculated hamster at intervals of 2, 4, and 6 weeks after inoculation. Blood cells were stained with Gienisa and examined for B. microti at x1,008 magnification, and the percentage of infected erythrocytes was recorded (10). Hamsters were judged to be negative for B. microti after no parasites were observed in 75 to 100 fields of stained erythrocytes. Attempted isolation of B. microti and B. burgdorferi from white-footed mice. White-footed mice were trapped in and near the yards of eight persons with babesiosis living in 3 southeastern Connecticut towns and in 15 other towns geographically scattered in Connecticut where murine surveillance for B. burgdorferi was already occurring. All mice except two, which were captured in 1988, were caught in 1989 and 1990 in Sherman box traps baited with peanut butter, apples, and sunflower seeds. Captured mice were returned to the laboratory, where attempts were made to isolate B. microti and B. burgdorferi from each animal (2). Cardiac blood from each mouse was drawn into a heparinized syringe, and 0.1 to 0.5 ml was inoculated intraperitoneally into one 3- to 9-week-old Syrian hamster. Microscopic examination of hamster blood smears for B. microti was done as described above. Attempts to isolate B. burgdorferi from each mouse were made by inoculating spleen, kidney, bladder, and skin tissues into Barbour-Stoenner-Kelly medium (1, 5, 17, 25, 27). Inoculated medium was kept at 32 C and was examined for borreliae by dark-field microscopy 4 to 6 weeks after inoculation. Spirochetes were identified as B. burgdorferi by their reaction with monoclonal antibody H5332 in an indirect fluorescent-antibody test (6). B. microti serology. Sera from humans diagnosed with babesiosis were tested for B. microti antibody by a standard indirect fluorescent-antibody test (12). Antigen was prepared from hamster erythrocytes parasitized with B. microti from a Connecticut white-footed mouse. Heparinized blood was drawn from the heart of a hamster with parasitemia of.30%. Following centrifugation of the blood with a Sorvall RC-5B centrifuge with an SM24 rotor at 2,000 rpm for 10 min and removal of the plasma, the erythrocytes were washed three times with 0.01 M phosphate-buffered saline (PBS; ph 7.6) by centrifugation at 2,000 rpm for 10 min. After the ceils were resuspended in PBS to a total of the original volume, antigen slides were prepared by dropping the antigen-cell suspension onto each of 12 wells on a slide (Cel-Line Associates, Newfield, N.J.). Following air drying, slides were wrapped in tissue paper and frozen at -60 C. Slides were used 1 to 30 weeks after being frozen. Slides were thawed when they were needed for indirect fluorescent-antibody testing. Human sera were diluted in 0.01 M PBS (ph 7.6), and fourfold dilutions were placed onto slides. Following an incubation period of 30 min at 37 C, slides were rinsed once in 0.01 M PBS (ph 7.6), washed in PBS in a shaker bath for 15 min, and allowed to dry. Goat anti-human immunoglobulin G (IgG) conjugate labeled with fluorescein isothiocyanate (GIBCO, Grand Island, N.Y.) diluted to 1:200 in 0.01 M PBS (ph 7.6) with 1.0% Evans Blue was applied to the wells, and the slides were reincubated for 30 min at 37 C. Slides were washed J. CLIN. MICROBIOL. three times in 0.01 M PBS (ph 7.6), allowed to dry, and then mounted in buffered glycerol mounting medium (BBL, Cockeysville, Md.). After being overlaid with a coverslip, each slide was examined with a Zeiss fluorescence microscope. Levels of fluorescence were scored on a scale of 1 to 4, with -3 being considered positive. Controls consisted of a known positive (high positive titer, 1:1,024) and a known negative (no fluorescence at all) serum specimen. B. burgdorferi serology. A whole-cell antigen enzymelinked immunosorbent assay was used to detect antibody to B. burgdorferi in humans (20). Cutoff optical density values of >0.21, >0.18, and.0.13 were considered positive for serum dilutions of 1:160, 1:320, and -1:640, respectively. Horseradish peroxidase-labeled polyvalent goat anti-human IgG (heavy and light chain specific) (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, Md.) and goat antihuman IgM (Fi-chain specific; Kirkegaard and Perry) were diluted 1:6,000 in PBS (ph 7.2) solution in the tests. RESULTS B. microti was isolated from 7 of 12 patients diagnosed with babesiosis (Table 1). Patients presented with fever ranging from 97.4 to F (36.3 to 39 C), headache, and fatigue. Two of nine patients remembered a prior tick bite. Erythrocytes infected with babesiosislike organisms were observed in stained smears of blood from 12 of the patients. With one exception, isolates were obtained from blood drawn from patients prior to and up to 4 days after treatment with clindamycin and quinine. One patient (patient 2) relapsed and was retreated. B. microti was isolated from blood drawn 39 days after initiation of the first treatment and 13 days before the second treatment. Of the five patients from whom B. microti was not isolated, one had been diagnosed 12 months before blood was drawn (patient 1) and the second one had been diagnosed 1.0 month before blood was drawn (patient 3). Neither patient had microscopically apparent parasitemia at the time. Clotted blood from a third patient (patient 13) was tested, and blood from a fourth patient (patient 6) was tested 19 days after treatment was initiated and after the patient received an exchange transfusion. B. microti was not isolated from a fifth patient (patient 4) from whom EDTAanticoagulated blood was drawn and tested within 1 day after initiation of treatment. Antibody titers to B. microti for 12 persons at the time of their clinical diagnosis with babesiosis ranged from 1:1,024 to 1:4,096 (Table 1). Blood from five of eight patients also had elevated antibody titers to B. burgdorferi (Table 1). B. microti was isolated from white-footed mice captured on or closely adjacent to the properties of eight patients diagnosed with babesiosis (Table 2). Of the 59 mice captured, 27 (46%) were infected with B. microti. B. burgdorferi was more prevalent and was isolated from 38 (64%) of the mice. Twenty-five mice (42%) were infected with both organisms. White-footed mice were captured in 18 towns in Connecticut, including the 3 towns in which case patients resided, and were tested for B. microti and B. burgdorferi (Table 3). B. burgdorferi was cultured from mice captured in 12 towns, and B. microti was recovered from mice captured in 6 towns. While most mice infected with B. microti were collected in southeastern Connecticut, one mouse infected with B. microti was collected in the north-central part of the state. Most of the mice were collected in 1989 and 1990, but two mice that were captured in 1988 in Lyme, Conn., and held in

3 VOL. 29, 1991 BABESIA MICROTI AND HUMAN BABESIOSIS 2781 TABLE 1. Demographic, treatment, outcome, and laboratory features of 13 patients with babesiosis, Connecticut, 1988 to 1990 Patient: Date Titers at time of (mo/day/yr): hospitalization to: Blood Treatment burgdorferi result Patient out- No.Sex Age Town of First hospi- Treatment drawn for B.smicroti (yr) residence talized initiated hamster inoculation IgG IgM 1 Male 84 Stonington 8/30/88 8/31/88 8/21/89 C+Qa 1:4,096 Negative Negative Negative Recoveredb 2 Male 71 Stonington 7/10/89' 7/13/89 8/21/89 C+Qd 1:1,024 1:1,280 Negative Positive Relapsed/ recovered 3 Male 86 Stonington 7/22/89 7/24/89 8/21/89 C+Q 1:4,096 1:5,120 1:5,120 Negative Recovered 4 Female 68 Stonington 8/14/89 8/15/89 8/16/89 C+Q 1:1,024 ND' ND Negative Recovered 5 Male 74 Old Lyme 8/17/89 None 8/21/89 None 1:1,024 ND ND Positive Recovered 6 Male 78 Stonington 8/02/89 8/02/89 8/21/89 C+Qf 1:1,024 ND ND Negative -g 7h Male 46 Waterbury 9/12/89 9/13/89 9/16/89 C+Q 1:1,024 Negative Negative Positive Recovered 8i Female 61 Old Lyme NW None 9/25/89 None 1:1,024k 1:2,560 Negative Positive Recovered 9 Male 82 Stonington 10/19/89 10/20/89 10/20/89 C+Q 1:4,096 Negative 1:1,280 Positive Recovered 10 Female 95 Stonington 7/06/90 7/07/90 7/08/90 C+Q 1:4,096 1:640 1:2,560 Positive Died 11 Female 66 Stonington 7/21/90 7/21/90 7/25/90 C+Q ND ND ND Positive Recovered 12 Male 70 Stonington 8/09/90 8/13/90 ND C+Q 1:1,024 ND ND ND Died 13' Male 88 Montville 8/18/90 8/20/90 8/18/90 C+-Q 1:1,024 Negative ND Negative Recovered C and Q. clindamycin and quinine, respectively. b Recovered is defined as the absence of B. tnicroti in erythrocytes of patients as determined by a 20-min examination of a blood smear stained with Wright's stain and resolution of symptoms, including headache, fever, and fatigue. Fatigue, however, often persisted for several weeks after discharge from the hospital. 'The patient was hospitalized twice (11 to 19 July 1989 and 6 to 14 September 1989). d The patient was treated twice (13 to 18 July 1989 and 3 to 21 September 1989). ND, not done. f- The patient also received an exchange transfusion. l The patient was discharged on 14 September 1989 without evidence of parasitemia, but he was readmitted on 16 September 1989 with chronic obstructive pulmonary disease and subsequently died. "Asplenic patient who acquired babesiosis by transfusion (22). Subclinical case; blood donor of transfusion-acquired babesiosis (22). NH, not hospitalized. Serum was obtained on the same date that blood was drawn for hamster inoculation. 'Clotted blood was inoculated into the hamster. captivity until 1989 were included in the analyses. One mouse was dually infected with B. microti and B. burgdorferi. Parasitemias in hamsters inoculated with infectious blood from humans and white-footed mice varied (Table 4). Of the seven isolates from humans, maximum parasitemias in hamsters varied from <5% to almost 50%. Parasitemias in hamsters from isolates obtained from 33 mice were often <5%, although parasitemia in one hamster was >50%. DISCUSSION B. microti is prevalent in white-footed mice in southeastern Connecticut and causes disease in humans. Heretofore, human babesiosis was assumed to be a disease that is contracted on islands (13, 16). All but four of the documented cases to date were reportedly acquired on the offshore islands of Massachusetts (Nantucket and Martha's Vineyard) or Rhode Island or on Long Island, Shelter Island, and Fire Island, N.Y. The 12 confirmed cases of hurnan babesiosis acquired by tick bite in Connecticut are the largest number reported for a non-island habitat. Furthermore, the prevalence of infected white-footed mice (.57%) at sites within three Connecticut towns approaches the prevalence levels reported for mice on islands in Massachusetts and Rhode Island and the peninsula of Cape Cod, Mass. (2, 3, 29). Babesiosis is a new tick-associated illness in Connecticut. B. microti was not isolated from white-footed mice during a TABLE 2. Prevalence of B. microti and B. burgdorferi in white-footed mice captured in or near the yards of patients diagnosed with human babesiosis, Connecticut, 1989 to 1990 Total no. (%) of mice Natient identifi- No. of miceninfected with: Patient identifi- Town B. microti and B. microti B. burgdorferi B. microti B. burgdorferi B. burgdorferi only only 2 Stonington (57) 6 (86) 3 Stonington 1 1 O O 1 (100) 1 (100) 5 Old Lyme 3 3 O 0 3(100) 3(100) 9 Stonington (7) 5 (36) 10 Stonington (86) 6 (86) il Stonington il O (9) 5 (45) 12 Stonington 10 6 O O 6 (60) 6 (60) 13 Montville 6 5 O 1 5 (83) 6 (100)

4 2782 ANDERSON ET AL. J. CLIN. MICROBIOL. TABLE 3. Prevalence of B. microti and B. burgdorferi in white-footed mice captured in 18 towns from five counties in Connecticut, 1988 to 1990' No. of mice No. of mice infected with: Total no. (%) of mice infected with: County and town tested B. microti and B. microti B. burgdorferi B. microti B. burgdorferi B. burgdorferi only only Hartford Burlington West Hartford (33) 0 Avon Windham, Killingly Fairfield Stamford (58) Westport Weston (50) Newtown Wilton (60) Middlesex Chester (33) East Haddam (12) 19 (59) Durham (13) New London Lyme (24) 9 (53) Old Lyme (60) 4 (80) Stonington (33) 33 (55) North Stonington (67) Montville (83) 6 (100) East Lyme (100) Total a Includes mice captured near the homes of patients diagnosed with babesiosis. statewide survey in 1976 and 1977, although antibodies were detected in nine mice (4). Antibodies in human sera were initially detected in blood samples that were drawn during 1986 (19). Our isolation of B. microti from a white-footed mouse captured in 1988 and the first reported human case that same year clearly document the presence of this pathogen in Connecticut. These clinical, serologic, and isolation data suggest to us that B. microti became prevalent in Connecticut in the mid to late 1980s. Although B. microti has been isolated from humans who reside in three southeastern Connecticut towns, our isolation of this pathogen from a white-footed mouse captured in West Hartford in north-central Connecticut indicates to us that B. microti may spread into other areas where I. dammini becomes established. Persons living in or visiting these new foci will need to be educated on ways of reducing the risk of tick bite and on the signs and symptoms of human babesiosis as well as those of Lyme disease. While the illness caused by B. microti is self-limited in the majority of cases, the two deaths among the 12 Connecticut cases acquired by tick bite TABLE 4. Maximum parasitemia in Syrian hamsters inoculated with B. microti from humans and white-footed mice Host origin of B. microti No. of isolates with maximum parasitemia (%) of: >50 Human White-footed mouse underscore the seriousness of this disease among elderly individuals (8). Babesiosis, like Lyme disease (14), is often a peridomestic disease. Mice captured near the homes of eight patients in Connecticut often had a high prevalence of parasitemia with B. microti. The majority of patients were more than 70 years old and had not visited outdoor areas other than their gardens or yards immediately prior to their illness. It is therefore likely that they acquired the infection following tick bites they obtained near their homes. Dual infections with B. microti and B. burgdorferi in many mice confirm that coinfection is common (2, 3). Relatively large numbers of I. dammini nymphs from islands where Lyme disease and babesiosis are prevalent also are simultaneously infected with both pathogens (24). These concurrent infections in reservoir hosts and tick vectors explain, in part, the relatively high percentage of patients diagnosed with Lyme disease who also have antibody to B. microti (7, 19, 23). Our finding of antibody to B. burgdorferi in five of eight patients with confirmed babesiosis suggests that dual infections are occurring in southeastern Connecticut. Our finding of highly variable maximum parasitemia in hamsters inoculated with infectious human or white-footed mouse blood confirms findings of earlier studies that courses of infection are variable (10, 34). Notably, the majority of our positive hamsters had -5% infected cells. The level of parasitemia of the inoculated blood and the elapsed time between blood collection and inoculation may be associated with the success of animal inoculation (10). The reason for our failure to isolate B. microti from one human with patent

5 VOL. 29, 1991 parasitemia is unknown. Prior antibiotic treatment of the patient may have contributed to our inability to isolate B. microti, although we successfully recovered it from three patients who had been treated with antibiotics. The use of splenectomized hamsters may enhance the success of isolating B. microti from blood with low-level parasitemia (9). Therefore, this method may be helpful when few or no parasites are detected on microscopic examination of blood smears from patients suspected of being infected. ACKNOWLEDGMENTS We thank Jim Ayers, Bonnie Hamid, and Niel Infante for technical assistance and James Hadler and Matthew Cartter for logistical support and manuscript review. Alan Barbour, University of Texas Health Science Center, San Antonio, provided the monoclonal antibodies. REFERENCES 1. Anderson, J. F., R. C. Johnson, L. A. Magnarelli, and F. W. Hyde Identification of endemic foci of Lyme disease: isolation of Borrelia burgdorferi from feral rodents and ticks (Dermacentor variabilis). J. Clin. Microbiol. 22: Anderson, J. F., R. C. Johnson, L. A. Magnarelli, F. W. Hyde, and J. E. Myers Peromyscus leucopus and Microtus pennsylvanicus simultaneously infected with Borrelia burgdorferi and Babesia microti. J. Clin. Microbiol. 23: Anderson, J. F., R. C. Johnson, L. A. Magnarelli, F. W. Hyde, and J. E. Myers Prevalence of Borrelia burgdorferi and Babesia microti in mice on islands inhabited by white-tailed deer. Apple. Environ. Microbiol. 53: Anderson, J. F., L. A. Magnarelli, and J. Kurz Intraerythrocytic parasites in rodent populations of Connecticut. Babesia and Grahamella species. J. Parasitol. 65: Barbour, A. G Isolation and cultivation of Lyme disease spirochetes. Yale J. Biol. Med. 57: Barbour, A. G., S. L. Tessier, and W. J. Todd Lyme disease spirochetes and ixodid tick spirochetes share a common surface antigenic determinant defined by a monoclonal antibody. Infect. Immun. 41: Benach, J. L., J. L. Coleman, G. S. Habicht, A. MacDonald, E. Grunwaldt, and J. A. Giron Serological evidence for simultaneous occurrences of Lyme disease and babesiosis. J. Infect. Dis. 152: Benach, J. L., and G. S. Habicht Clinical characteristics of human babesiosis. J. Infect. Dis. 144: Benach, J. L., D. J. White, and J. P. McGovern Babesiosis in Long Island, host-parasite relationships of rodent- and human-derived Babesia microti isolates in hamsters. Am. J. Trop. Med. Hyg. 27: Brandt, F., G. R. Healy, and M. Welch Human babesiosis: the isolation of Babesia microti in golden hamsters. J. Parasitol. 63: Burgdorfer, W., A. G. Barbour, S. F. Hayes, J. L. Benach, E. Grunwaldt, and J. P. Davis Lyme disease-a tick borne spirochetosis? Science 216: Chisholm, E. S., T. K. Ruebush, A. J. Suizer, and G. R. Healy Babesia microti infection in man: evaluation of an indirect immunofluorescent antibody test. Am. J. Trop. Med. Hyg. 27: Dammin, G. J., A. Spielman, J. L. Benach, and J. Piesman The rising incidence of clinical Babesia microti infection. Hum. Pathol. 12: Falco, R. C., and D. Fish Prevalence of Ixodes dammini near the homes of Lyme disease patients in Westchester County, New York. Am. J. Epidemiol. 127: Gadbaw, J. J., J. F. Anderson, M. L. Cartter, and J. L. Hadler. BABESIA MICROTI AND HUMAN BABESIOSIS Babesiosis-Connecticut. Morbid. Mortal. Weekly Rep. 38: Healy, G., and M. Ristic Human babesiosis, p In M. Ristic (ed.), Babesiosis of domestic animals and man. CRC Press, Inc., Boca Raton, Fla. 17. Johnson, R. C., N. Marek, and C. Kodner Infection of Syrian hamsters with Lyme disease spirochetes. J. Clin. Microbiol. 20: Johnson, R. C., G. P. Schmid, F. W. Hyde, A. G. Steigerwalt, and D. J. Brenner Borrelia burgdorferi sp. nov.: etiologic agent of Lyme disease. Int. J. Syst. Bacteriol. 34: Krause, P. J., S. R. Telford HII, R. Ryan, A. B. Hurta, I. Kwasnik, S. Luger, J. Niederman, M. Gerber, and A. Spielman Geographical and temporal distribution of babesial infection in Connecticut. J. Clin. Microbiol. 29: Magnarelli, L. A., J. M. Meegan, J. F. Anderson, and W. A. Chappell Comparison of an indirect fluorescent-antibody test with an enzyme-iinked immunosorbent assay for serological studies of Lyme disease. J. Clin. Microbiol. 20: Miller, G. L., R. B. Craven, R. E. Bailey, and T. F. Tsai The epidemiology of Lyme disease in the United States Lab. Med. 21: Mintz, E. D., J. F. Anderson, R. G. Cable, and J. L. Hadler Transfusion-transmitted babesiosis: a case report from a new endemic area. Transfusion 31: Mintz, E. D., J. F. Anderson, J. L. Hadler, and M. L. Cartter Cluster of babesiosis in Connecticut, Epidemic Intelligence Service Conference Abstracts, April 1990, p Piesman, J., T. N. Mather, S. R. Telford III, and A. Spielman Concurrent Borrelia burgdorferi and Babesia microti infection in nymphal Ixodes dammini. J. Clin. Microbiol. 24: Schwan, T. G., W. Burgdorfer, M. E. Schrumpf, and R. H. Karstens The urinary bladder, a consistent source of Borrelia burgdorferi in experimentally infected white-footed mice (Peromyscus leucopus). J. Clin. Microbiol. 26: Scrimenti, R. C Erythema chronicum migrans. Arch. Dermatol. 102: Sinsky, R. J., and J. Piesman Earpunch biopsy method for detection and isolation of Borrelia burgdorferi from rodents. J. Clin. Microbiol. 27: Spielman, A Human babesiosis on Nantucket Island: transmission by nymphal Ixodes ticks. Am. J. Trop. Med. Hyg. 25: Spielman, A., P. Etkind, J. Piesman, T. K. Ruebush 11, D. D. Juranek, and M. S. Jacobs Reservoir hosts of human babesiosis on Nantucket Island. Am. J. Trop. Med. Hyg. 30: Steere, A. C Lyme disease. N. Engl. J. Med. 312: Steere, A. C., and S. E. Malawista Cases of Lyme disease in the United States: locations correlated with distribution of Ixodes dammini. Ann. Intern. Med. 91: Steere, A. C., S. E. Malawista, D. R. Snydman, R. E. Shope, W. A. Andiman, M. R. Ross, and F. M. Steele Lyme arthritis: an epidemic of oligoarticular arthritis in children and adults in three Connecticut communities. Arthritis Rheum. 20: Steketee, R. W., M. R. Eckman, E. C. Burgess, J. N. Kuritsky, J. Dickerson, W. L. Schell, M. S. Godsey, and J. P. Davis Babesiosis in Wisconsin, a new focus of disease transmission. J. Am. Med. Assoc. 253: Van Peenen, P. F. D., and G. R. Healy Infection of Microtus ochrogaster with piroplasms isolated from man. J. Parasitol. 56: Western, K. A., G. D. Benson, N. N. Gleason, G. R. Healy, and M. G. Schultz Babesiosis in a Massachusetts resident. N. Engl. J. Med. 283: