JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1991, p. 2779-27830095-1137/91/122779-05$02.00/0Copyright C 1991, American Society for Microbiology

Vol. 29, No. 12

Babesia microti, Human Babesiosis, and Borrelia burgdorferiin 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 065041;Epidemiology Section, Connecticut Department of Health Services, Hartford, Connecticut 06106, and Division of

Field Epidemiology, Epidemiology Program Office, Centers for Disease Control, Atlanta, Georgia 303332;and Lawrence and Memorial Hospital, New London, Connecticut 063203

Received 10 June 1991/Accepted 13 September 1991

Babesia microti was isolated from a white-footed mouse (Peromyscus leucopus) that was captured insoutheastern 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 humancases reported on the mainland United States. Two of nine patients queried remembered a prior tick bite. SinceBabesia parasites are known to be vectored only by ticks, we surmise that 12 of these infections were acquiredvia tick bites; 1 was obtained by blood transfusion (the patient was 46 years of age) from an endemically infecteddonor. The ages of the patients with tick-acquired babesiosis ranged from 61 to 95 years. Two patients died withactive infections, and one patient died from chronic obstructive pulmonary disease soon after treatment withclindamycin and quinine. Indirect fluorescent-antibody titers of blood samples drawn at the time ofhospitalization for 11 patients and at the time of active infection for 1 asymptomatic person ranged from1:1,024 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 hamstersinoculated with blood from 7 of 12 patients tested and was also isolated from mice captured in six towns. Theperidomestic nature of the disease was demonstrated by isolating the parasite from white-footed mice capturedin or near the yards of eight different patients. Of 59 mice tested, 27 were positive and 25 were coinfected withB. 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 thispathogen may spread into other areas where Ixodes dammini, the tick vector, becomes established.

Two new tick-associated illnesses were reported fromwidely different geographic regions of the United States in1970. Erythema chronicum migrans, now known as Lymedisease (32), was reported in Wisconsin (26) and humanbabesiosis was documented on Nantucket Island, Mass.(35). Lyme disease is a multisystemic illness affecting joint,heart, skin, nervous system, and other tissues (30), whilebabesiosis is a malarialike illness that often results in head-ache, malaise, anemia, and fatigue (35). The two etiologicagents, Borrelia burgdorferi (11, 18) and Babesia microti(35), were subsequently demonstrated to be transmitted bythe same species of tick, Ixodes dammini (11, 28, 31).

While B. burgdorferi has become the most common vec-tor-borne pathogen in the United States, with 4,572 casesbeing reported from 43 states in 1988 (21), less than 250 casesof babesiosis caused by B. microti are known (16). Almost allof the tick-associated cases of babesiosis have been acquiredon islands in the northeastern United States. Exceptionsinclude two cases reported in Wisconsin (33) and two casesdocumented on Cape Cod, Mass. (13).

In 1988, a possible new endemic focus of B. microti on themainland of the United States was recognized when a singleclinical case of babesiosis was reported in a Stonington,Conn., resident with no recent travel history. In the summerof 1989, four additional cases were reported in residents ofthe same town, and a fifth case was reported in a resident ofOld Lyme, Conn. (15). By December 1989, a total of ninecases 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 toB. microti. In one study (19), 9.5% of persons tested state-wide had elevated antibody titers (21:64) to B. microti. Inanother study (23), 37% of persons tested who lived in thesame towns as patients with clinical babesiosis had elevatedtiters (21:64) to B. microti.We now report (i) the largest number of babesiosis cases

to date that have been contracted on the mainland UnitedStates, (ii) the isolation of B. microti from humans living insoutheastern Connecticut, (iii) the isolation of B. microtifrom white-footed mice (Peromyscus leucopus) captured inor near the yards of humans diagnosed with babesiosis insoutheastern Connecticut and from woodlands in southeast-ern and north-central Connecticut, (iv) the isolation of B.microti from a mouse captured in Connecticut as early as1988, and (v) a relatively high prevalence of white-footedmice dually infected with B. microti and B. burgdorferi.

MATERIALS AND METHODS

Attempted isolation of B. microti from humans. Blooddrawn from 11 patients suspected of infection with B. microtiwas placed into EDTA tubes, and blood drawn from onepatient with babesiosis was placed into a tube withoutanticoagulant. Blood smears from 12 patients were stainedwith Wright's stain and examined microscopically for Babe-sia parasites. The time that blood was drawn for attemptedisolation of Babesia parasites relative to the time of illness inthe 12 patients was variable, ranging from the first day ofhospitalization to almost 1 year after the onset of disease. A

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total of 0.5 to 1 ml of uncoagulated blood from each personwas inoculated intraperitoneally into one 3- to 6-week-oldSyrian hamster for attempted isolation of B. microti (10).The clotted blood sample was shaken, and clumps of eryth-rocytes were drawn into a syringe. A total of 0.2 ml ofclotted blood and serum was inoculated intraperitoneallyinto one hamster. Blood smears were obtained from tailsnips from each inoculated hamster at intervals of 2, 4, and6 weeks after inoculation. Blood cells were stained withGienisa and examined for B. microti at x1,008 magnifica-tion, and the percentage of infected erythrocytes was re-corded (10). Hamsters were judged to be negative for B.microti after no parasites were observed in 75 to 100 fields ofstained erythrocytes.Attempted isolation of B. microti and B. burgdorferi from

white-footed mice. White-footed mice were trapped in andnear the yards of eight persons with babesiosis living in 3southeastern Connecticut towns and in 15 other townsgeographically scattered in Connecticut where murine sur-veillance for B. burgdorferi was already occurring. All miceexcept two, which were captured in 1988, were caught in1989 and 1990 in Sherman box traps baited with peanutbutter, apples, and sunflower seeds. Captured mice werereturned to the laboratory, where attempts were made toisolate B. microti and B. burgdorferi from each animal (2).Cardiac blood from each mouse was drawn into a heparin-ized syringe, and 0.1 to 0.5 ml was inoculated intraperitone-ally into one 3- to 9-week-old Syrian hamster. Microscopicexamination of hamster blood smears for B. microti wasdone as described above.Attempts to isolate B. burgdorferi from each mouse were

made by inoculating spleen, kidney, bladder, and skin tis-sues into Barbour-Stoenner-Kelly medium (1, 5, 17, 25, 27).Inoculated medium was kept at 32°C and was examined forborreliae by dark-field microscopy 4 to 6 weeks after inocu-lation. Spirochetes were identified as B. burgdorferi by theirreaction with monoclonal antibody H5332 in an indirectfluorescent-antibody test (6).

B. microti serology. Sera from humans diagnosed withbabesiosis were tested for B. microti antibody by a standardindirect fluorescent-antibody test (12). Antigen was preparedfrom hamster erythrocytes parasitized with B. microti from aConnecticut white-footed mouse. Heparinized blood wasdrawn from the heart of a hamster with parasitemia of.30%. Following centrifugation of the blood with a SorvallRC-5B centrifuge with an SM24 rotor at 2,000 rpm for 10 minand removal of the plasma, the erythrocytes were washedthree times with 0.01 M phosphate-buffered saline (PBS; pH7.6) by centrifugation at 2,000 rpm for 10 min. After the ceilswere resuspended in PBS to a total of the original volume,antigen slides were prepared by dropping the antigen-cellsuspension onto each of 12 wells on a slide (Cel-LineAssociates, Newfield, N.J.). Following air drying, slideswere wrapped in tissue paper and frozen at -60°C. Slideswere used 1 to 30 weeks after being frozen.

Slides were thawed when they were needed for indirectfluorescent-antibody testing. Human sera were diluted in0.01 M PBS (pH 7.6), and fourfold dilutions were placedonto slides. Following an incubation period of 30 min at37°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 todry. Goat anti-human immunoglobulin G (IgG) conjugatelabeled with fluorescein isothiocyanate (GIBCO, GrandIsland, N.Y.) diluted to 1:200 in 0.01 M PBS (pH 7.6) with1.0% Evans Blue was applied to the wells, and the slideswere reincubated for 30 min at 37°C. Slides were washed

three times in 0.01 M PBS (pH 7.6), allowed to dry, and thenmounted in buffered glycerol mounting medium (BBL,Cockeysville, Md.). After being overlaid with a coverslip,each slide was examined with a Zeiss fluorescence micro-scope. Levels of fluorescence were scored on a scale of 1 to4, with -3 being considered positive. Controls consisted ofa known positive (high positive titer, 1:1,024) and a knownnegative (no fluorescence at all) serum specimen.B. burgdorferi serology. A whole-cell antigen enzyme-

linked immunosorbent assay was used to detect antibody toB. burgdorferi in humans (20). Cutoff optical density valuesof >0.21, >0.18, and .0.13 were considered positive forserum dilutions of 1:160, 1:320, and -1:640, respectively.Horseradish peroxidase-labeled polyvalent goat anti-humanIgG (heavy and light chain specific) (Kirkegaard and PerryLaboratories, Inc., Gaithersburg, Md.) and goat antihumanIgM (Fi-chain specific; Kirkegaard and Perry) were diluted1:6,000 in PBS (pH 7.2) solution in the tests.

RESULTS

B. microti was isolated from 7 of 12 patients diagnosedwith babesiosis (Table 1). Patients presented with feverranging from 97.4 to 102.4°F (36.3 to 39°C), headache, andfatigue. Two of nine patients remembered a prior tick bite.Erythrocytes infected with babesiosislike organisms wereobserved in stained smears of blood from 12 of the patients.With one exception, isolates were obtained from blooddrawn from patients prior to and up to 4 days after treatmentwith clindamycin and quinine. One patient (patient 2) re-lapsed and was retreated. B. microti was isolated from blooddrawn 39 days after initiation of the first treatment and 13days before the second treatment.Of the five patients from whom B. microti was not

isolated, one had been diagnosed 12 months before bloodwas drawn (patient 1) and the second one had been diag-nosed 1.0 month before blood was drawn (patient 3). Neitherpatient had microscopically apparent parasitemia at thetime. Clotted blood from a third patient (patient 13) wastested, and blood from a fourth patient (patient 6) was tested19 days after treatment was initiated and after the patientreceived an exchange transfusion. B. microti was not iso-lated from a fifth patient (patient 4) from whom EDTA-anticoagulated blood was drawn and tested within 1 day afterinitiation of treatment.Antibody titers to B. microti for 12 persons at the time of

their clinical diagnosis with babesiosis ranged from 1:1,024to 1:4,096 (Table 1). Blood from five of eight patients alsohad elevated antibody titers to B. burgdorferi (Table 1).

B. microti was isolated from white-footed mice capturedon or closely adjacent to the properties of eight patientsdiagnosed with babesiosis (Table 2). Of the 59 mice cap-tured, 27 (46%) were infected with B. microti. B. burgdorferiwas more prevalent and was isolated from 38 (64%) of themice. Twenty-five mice (42%) were infected with bothorganisms.

White-footed mice were captured in 18 towns in Connect-icut, 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 insoutheastern Connecticut, one mouse infected with B. mi-croti 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

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TABLE 1. Demographic, treatment, outcome, and laboratory features of 13 patients with babesiosis, Connecticut, 1988 to 1990

Patient: Date (mo/day/yr): Titers at time ofhospitalization to:

Blood Treatment B. burgdorferi result Patient out-No.Sex Age Town of First hospi- Treatment drawn for B.smicroti

(yr) residence talized initiated hamsterinoculation IgG IgM

1 Male 84 Stonington 8/30/88 8/31/88 8/21/89 C+Qa 1:4,096 Negative Negative Negative Recoveredb2 Male 71 Stonington 7/10/89' 7/13/89 8/21/89 C+Qd 1:1,024 1:1,280 Negative Positive Relapsed/

recovered3 Male 86 Stonington 7/22/89 7/24/89 8/21/89 C+Q 1:4,096 1:5,120 1:5,120 Negative Recovered4 Female 68 Stonington 8/14/89 8/15/89 8/16/89 C+Q 1:1,024 ND' ND Negative Recovered5 Male 74 Old Lyme 8/17/89 None 8/21/89 None 1:1,024 ND ND Positive Recovered6 Male 78 Stonington 8/02/89 8/02/89 8/21/89 C+Qf 1:1,024 ND ND Negative -g7h Male 46 Waterbury 9/12/89 9/13/89 9/16/89 C+Q 1:1,024 Negative Negative Positive Recovered8i Female 61 Old Lyme NW None 9/25/89 None 1:1,024k 1:2,560 Negative Positive Recovered9 Male 82 Stonington 10/19/89 10/20/89 10/20/89 C+Q 1:4,096 Negative 1:1,280 Positive Recovered10 Female 95 Stonington 7/06/90 7/07/90 7/08/90 C+Q 1:4,096 1:640 1:2,560 Positive Died11 Female 66 Stonington 7/21/90 7/21/90 7/25/90 C+Q ND ND ND Positive Recovered12 Male 70 Stonington 8/09/90 8/13/90 ND C+Q 1:1,024 ND ND ND Died13' 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. Onemouse was dually infected with B. microti and B. burgdor-feri.

Parasitemias in hamsters inoculated with infectious bloodfrom humans and white-footed mice varied (Table 4). Of theseven isolates from humans, maximum parasitemias in ham-sters varied from <5% to almost 50%. Parasitemias inhamsters from isolates obtained from 33 mice were often<5%, although parasitemia in one hamster was >50%.

DISCUSSIONB. microti is prevalent in white-footed mice in southeast-

ern 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 docu-mented cases to date were reportedly acquired on theoffshore islands of Massachusetts (Nantucket and Martha'sVineyard) or Rhode Island or on Long Island, Shelter Island,and Fire Island, N.Y. The 12 confirmed cases of hurnanbabesiosis acquired by tick bite in Connecticut are thelargest number reported for a non-island habitat. Further-more, the prevalence of infected white-footed mice (.57%)at sites within three Connecticut towns approaches theprevalence levels reported for mice on islands in Massachu-setts 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 withhuman babesiosis, Connecticut, 1989 to 1990

Total no. (%) of miceNatientidentifi-No. of miceNinfected with:

Patient identifi- Town B. microti and B. microti B. burgdorferi B. microti B. burgdorferiB. burgdorferi only only

2 Stonington 7 4 0 2 4 (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 14 1 0 4 1 (7) 5 (36)10 Stonington 7 5 1 1 6 (86) 6 (86)il Stonington il O 1 5 1(9) 5 (45)12 Stonington 10 6 O O 6 (60) 6 (60)13 Montville 6 5 O 1 5 (83) 6 (100)

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TABLE 3. Prevalence of B. microti and B. burgdorferi in white-footed mice captured in 18 towns from five countiesin Connecticut, 1988 to 1990'

No. of mice infected with: Total no. (%) of mice infected with:County and town No. of mice

tested B. microti and B. microti B. burgdorferi B. microti B. burgdorferiB. burgdorferi only only

HartfordBurlington 1 0 0 0 0 0West Hartford 3 0 1 0 1 (33) 0Avon 9 0 0 0 0 0

Windham, Killingly 4 0 0 0 0 0

FairfieldStamford 12 0 0 7 0 7 (58)Westport 1 0 0 0 0 0Weston 2 0 0 1 0 1(50)Newtown 28 0 0 0 0 0Wilton 5 0 0 3 0 3 (60)

MiddlesexChester 3 0 0 1 0 1(33)East Haddam 32 4 0 15 4 (12) 19 (59)Durham 8 0 0 1 0 1(13)

New LondonLyme 17 3 1 6 4 (24) 9 (53)Old Lyme 5 3 0 1 3 (60) 4 (80)Stonington 60 17 3 16 20 (33) 33 (55)North Stonington 6 0 0 4 0 4 (67)Montville 6 5 0 1 5 (83) 6 (100)East Lyme 2 0 0 2 0 2 (100)

Total 204 32 5 58 37 90

a Includes mice captured near the homes of patients diagnosed with babesiosis.

statewide survey in 1976 and 1977, although antibodies weredetected in nine mice (4). Antibodies in human sera wereinitially detected in blood samples that were drawn during1986 (19). Our isolation of B. microti from a white-footedmouse captured in 1988 and the first reported human casethat same year clearly document the presence of this patho-gen in Connecticut. These clinical, serologic, and isolationdata suggest to us that B. microti became prevalent inConnecticut in the mid to late 1980s.Although B. microti has been isolated from humans who

reside in three southeastern Connecticut towns, our isolationof this pathogen from a white-footed mouse captured in WestHartford in north-central Connecticut indicates to us that B.microti may spread into other areas where I. damminibecomes established. Persons living in or visiting these newfoci will need to be educated on ways of reducing the risk oftick bite and on the signs and symptoms of human babesiosisas well as those of Lyme disease. While the illness caused byB. microti is self-limited in the majority of cases, the twodeaths among the 12 Connecticut cases acquired by tick bite

TABLE 4. Maximum parasitemia in Syrian hamsters inoculatedwith B. microti from humans and white-footed mice

No. of isolates with maximumHost origin of parasitemia (%) of:B. microti

.5 6-15 16-25 26-50 >50

Human 2 2 1 2 0White-footed mouse 17 2 8 5 1

underscore the seriousness of this disease among elderlyindividuals (8).

Babesiosis, like Lyme disease (14), is often a peridomesticdisease. Mice captured near the homes of eight patients inConnecticut often had a high prevalence of parasitemia withB. microti. The majority of patients were more than 70 yearsold and had not visited outdoor areas other than theirgardens or yards immediately prior to their illness. It istherefore likely that they acquired the infection followingtick bites they obtained near their homes.Dual infections with B. microti and B. burgdorferi in many

mice confirm that coinfection is common (2, 3). Relativelylarge numbers of I. dammini nymphs from islands whereLyme disease and babesiosis are prevalent also are simulta-neously infected with both pathogens (24). These concurrentinfections in reservoir hosts and tick vectors explain, in part,the relatively high percentage of patients diagnosed withLyme disease who also have antibody to B. microti (7, 19,23). Our finding of antibody to B. burgdorferi in five of eightpatients with confirmed babesiosis suggests that dual infec-tions are occurring in southeastern Connecticut.Our finding of highly variable maximum parasitemia in

hamsters inoculated with infectious human or white-footedmouse blood confirms findings of earlier studies that coursesof infection are variable (10, 34). Notably, the majority ofour positive hamsters had -5% infected cells. The level ofparasitemia of the inoculated blood and the elapsed timebetween blood collection and inoculation may be associatedwith the success of animal inoculation (10). The reason forour failure to isolate B. microti from one human with patent

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parasitemia is unknown. Prior antibiotic treatment of thepatient may have contributed to our inability to isolate B.microti, although we successfully recovered it from threepatients who had been treated with antibiotics. The use ofsplenectomized hamsters may enhance the success of isolat-ing B. microti from blood with low-level parasitemia (9).Therefore, this method may be helpful when few or noparasites are detected on microscopic examination of bloodsmears from patients suspected of being infected.

ACKNOWLEDGMENTS

We thank Jim Ayers, Bonnie Hamid, and Niel Infante for techni-cal assistance and James Hadler and Matthew Cartter for logisticalsupport and manuscript review. Alan Barbour, University of TexasHealth Science Center, San Antonio, provided the monoclonalantibodies.

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