Vol. 29, No. 12JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1991, p. 2798-28040095-1137/91/122798-07$02.00/0Copyright © 1991, American Society for Microbiology

Rickettsiae and Borrelia burgdorferi in Ixodid TicksLOUIS A. MAGNARELLI,1* THEODORE G. ANDREADIS,1 KIRBY C. STAFFORD III,'

AND CYNTHIA J. HOLLAND2t

Department ofEntomology, Connecticut Agricultural Experiment Station, P.O. Box 1106, New Haven,Connecticut 06504,1 and Hemotropic Disease Section, Department of Veterinary Pathobiology,

University of Illinois, Urbana, Illinois 618012

Received 13 June 1991/Accepted 26 September 1991

Nymphs and adults of hard-bodied ticks were collected in Connecticut and tested by direct and indirectimmunofluorescence staining methods for rickettsiae and Borrelia burgdorferi. Of the 609 Ixodes dammini ticksexamined, 59 (9.7%) harbored rickettsialike microorganisms in hemocytes (blood cells). These bacteria reactedwith fluorescein-conjugated antiserum to Ehrlichia canis, the etiologic agent of canine ehrlichiosis. Prevalenceof infection ranged from 6.8 to 12.7% for males and females, respectively. Although the specific identities ofthe hemocytic rickettsialike organisms are unknown, they share antigens with ehrlichiae. Electron microscopyrevealed rickettsiae in ovarian tissues of I. dammini that also had infected hemocytes. Rickettsialike organismswere also observed in the hemocytes of 5 (6.9%) of 73 Dermacentor variabiis ticks. In analyses for B. burg-dorferi, 146 (23.7%) of 617 I. dammini ticks harbored these spirochetes in midguts. Hemocytic rickettsialikemicroorganisms coexisted with B. burgdorferi in 36 (6.7%) of the 537 nymphs and adults of I. damminiexamined. J. dammini, with its broad host range, has the potential to acquire multiple microorganisms.

Public awareness of tick-borne pathogens and the diseasesthey cause is rising. The association between American dogticks, Dermacentor variabilis, and Rocky Mountain spottedfever is well recognized (9, 27). During the 1970s, increasedpopulations of Ixodes dammini and the occurrence of babe-siosis were reported (5, 17, 39). The subsequent discovery ofBorrelia burgdorferi, the causative agent of Lyme borreliosis(10, 43), in I. dammini and the apparent widespread geo-graphic distribution of this disease on different continents(40, 41) greatly elevated scientific interest in tick-relatedzoonotic diseases, particularly in North America and Eu-rope. More recently, there is evidence of yet another tick-associated human disease-ehrlichiosis (26). Human ehrli-chiosis has been reported in at least 16 states (12, 26, 36).Although the etiologic agent has not been isolated fromticks, it is believed to be Ehrlichia canis or a closely relatedorganism (14, 44). The brown dog tick, Rhipicephalus san-guineus, has been shown to be a vector of E. canis for dogs(20), but this tick rarely bites humans. Thus, the vector(s) ofhuman ehrlichiosis is unknown.During an epidemiological investigation ofhuman ehrlichi-

osis among army reservists (31), some patients recalled beingbitten by "tiny ticks." The tick species was not identified, butthe training exercises were conducted in areas of New Jerseywhere I. dammini, Amblyomma americanum (lone star ticks),and Lyme borreliosis occur. Moreover, reports of human andcanine ehrlichiosis in southern states coincide with the distri-bution of Ixodes scapularis, a tick that is closely related to I.dammini and Ixodes ricinus. As a group, these ticks have awide host range that includes mammals, birds, and evenreptiles (1). The purpose of this study was to determinewhether I. dammini nymphs and adults and other ixodid(hard-bodied) ticks harbor rickettsiae and whether these mi-croorganisms coexist with B. burgdorferi.

* Corresponding author.t Present address: Protatek Reference Laboratory, Chandler, AZ

85225.

MATERIALS AND METHODS

Tick collection. Adults of I. dammini and D. variabiliswere collected mainly by dragging flannel cloth over vege-tation in or near woodlands during the period from 1989 to1991. Additional specimens were obtained from white-taileddeer (Odocoileus virginianus) killed during fall hunting sea-sons, woodchucks (Marmota monax) captured in Toma-hawk traps, and people who had detached ticks from them-selves. Nymphs of I. dammini and D. variabilis were alsoremoved from white-footed mice (Peromyscus leucopus) andchipmunks (Tamias striatus) caught in Sherman box traps(2). Other nymphs of I. dammini were removed from passe-rine birds caught in Japanese mist nets. The woodchucks andother rodents were released unharmed into their naturalhabitats following tick removal and recovery from anesthe-sia. Birds were examined without the use of anesthetics andwere likewise released unharmed after examination. I. dam-mini and D. variabilis were collected in 25 towns in Con-necticut, including East Haddam, Chester, Lyme, OldLyme, and Waterford, communities in southeastern andsouth-central parts of the state where Lyme borreliosis ishighly endemic (32).

Preparation of tick cells and tissues. Hemolymph (blood)and midgut tissues were obtained from living ticks andprocessed for serologic testing. Hemolymph was obtainedfrom ticks by amputating a leg (8) and then allowed to air dryon glass microscope slides. Midgut tissues were removedfrom ticks, and impression smears were prepared as re-ported earlier (10). All preparations were then air dried at37°C for at least 6 h and fixed in cold acetone for 10 min. Ifcell and tissue preparations could not be stained by immu-nofluorescence methods within 24 h, the slides were storedat -60°C until analyzed.FA staining. Direct fluorescent antibody (FA) staining

methods were primarily used to detect ehrlichiae in tickhemocytes. At the University of Illinois, dogs with nocross-reactive antibodies prior to immunization were inocu-lated with E. canis. When homologous antibody titers to E.canis reached 1:5,120 or greater, as determined by indirect

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FA staining, serum was obtained. Details on inoculationsand antiserum production, including purification of the im-munoglobulin G (IgG) antibody fraction by sodium sulfateprecipitation procedures, have been described previously(29). These antibodies were conjugated with fluoresceinisothiocyanate (FITC). This stock FA reagent was diluted1:30 in phosphate-buffered saline (PBS) (pH = 7.2), appliedto tick hemocyte preparations on glass microscope slides,incubated at 37°C for 30 min, washed twice in PBS solution(pH = 7.2), and then washed once in distilled water. Slideswere then mounted with buffered glycerol and coverslips andwere examined by fluorescence microscopy. In our tests ofcontrols, there was no reactivity of the direct FA reagentwith uninfected canine monocytes. Therefore, the anti-E.canis serum was not an antimonocytic serum and adsorptionwith uninfected canine monocytes was unnecessary.

Indirect FA staining of tick hemocytes was used to con-

firm direct FA staining results. However, there was insuffi-cient hemolymph from any given tick to routinely performboth tests in parallel experiments. Therefore, indirect FAstaining was usually applied to hemolymph preparations ofticks collected from the same populations included in studiesfor which direct FA staining methods were used. For indi-rect FA staining, the same unlabeled canine antiserum,having a homologous antibody titer of 1:10,240 to E. canis,was diluted in PBS to 1:64 before application to tick cellpreparations. Following a 30-min incubation period andwashes with PBS and distilled water, polyvalent FITC-labeled goat anti-dog IgG antibodies (specific for heavy andlight chains; Cooper Biomedical, Malvern, Pa.) were dilutedin PBS solution to 1:40, applied to the slide, incubated, andwashed as described above.

Seventy tick hemocyte preparations were tested withconvalescent-human serum by indirect FA staining. Theserum sample was from a person who was clinically diag-nosed as having ehrlichiosis. The titer of the antibody to E.canis was 1:10,240. This sample, also obtained from theUniversity of Illinois, was used at a working dilution (inPBS) of 1:80; polyvalent FITC-conjugated goat anti-humanimmunoglobulin (GIBCO Laboratories, Grand Island,N.Y.), diluted to 1:40, were added as a second antibody.Duplicate preparations of hemolymph were also tested byindirect FA staining with a monoclonal antibody to Ehrlichiaspecies (134-5B1). The production and use of this genus-

specific antiserum, which reacted with a 57- to 58-kDaantigen of Ehrlichia species, has been reported (13).

Detection of B. burgdorferi in tick midgut tissues was

likewise accomplished by indirect FA staining. Murinemonoclonal antibodies (H5332), directed to outer surfaceprotein A of B. burgdorferi, and a 1:60 dilution of FITC-conjugated goat anti-mouse IgG (specific for heavy and lightchains) antibodies (Organon Teknika Corp., Durham, N.C.)were used as previously reported (22).

Specificity tests. Indirect FA staining methods were ap-plied to assess serologic cross-reactivity between the Ehrli-chia spp. and related microorganisms. The canine andhuman sera, with antibodies to E. canis, were tested againstE. canis (infected canine monocytes were used as theantigen) in homologous tests and against the following puri-fied spotted fever group (SFG) rickettsiae: Rickettsia rick-ettsii (R strain), Rickettsia montana (M15-6), and Rickettsiarhipicephali (3-7 9 -6). Formalin-treated antigens of theseSFG rickettsiae were prepared at the Rocky MountainLaboratories (Hamilton, Mont.). Details of the preparationof standardized concentrations of stock rickettsial antigens,stabilization procedures, and use of antigens in serologic

TABLE 1. Examination of nymphs and adults of I. dammminitested for rickettsialike microorganisms and B. burgdorferi

Rickettsialike organisms" B. burgdorferibYear and tickstage or sex Total no. of % Positive Total no. of % Positive

ticks tested ticks tested

1989Nymph 194 12.4 195 13.9Male 158 5.7 85 32.9Female 98 7.1 173 20.0

1990Nymph 21 0 21 4.8Male 78 9.0 80 37.5Female 60 21.7 63 36.5

Total 609 9.7 617 23.0

aExamination of tick hemocytes by direct or indirect FA staining withantiserum to E. canis. Note that positive reactions indicate shared antigenswith ehrlichiae.

b Examination for B. burgdorferi found in tick midgut tissues.

testing have been published previously (23, 24, 30, 33). Thedirect FA reagent for E. canis was tested against Wolbachiapersica (a rickettsial symbiote), Ehrlichia risticii (the caus-ative agent of Potomac horse fever), the SFG antigens, andE. canis. Monoclonal antibodies to W. persica (134-5C9) andto Ehrlichia species (134-5B1) were included to verify anti-gen reactivity in homologous tests.

Sera from six patients who had clinical manifestations ofRocky Mountain spotted fever were tested by indirect FAstaining methods against E. canis, the SFG rickettsiae, W.persica, and hemocytes of I. dammini. Titers of antibody toR. rickettsii ranged from 1:4,096 to 1:16,284 by indirect micro-immunofluorescence staining (33). Sera were obtained frompersons in Connecticut from 1980 to 1983 and had been storedat -60°C at the Connecticut Agricultural Experiment Station.

Electron microscopy. Tissues from unfed female I. dam-mini that had rickettsialike microorganisms in hemocyteswere dissected and were fixed for 2 h at room temperature(22 to 24°C) in a solution consisting of 2.5% glutaraldehyde,2% paraformaldehyde, 0.1% CaCl2, and 1% sucrose, buff-ered with 0.1 M sodium cacodylate (pH = 7.2). Specimenswere postfixed for 2 h at room temperature in 1% OSO4 in thesame buffer, stained en bloc with 2% uranyl acetate, rapidlydehydrated through an ethanol-and-acetone series, and em-bedded in an LX 112-araldite mixture. Ultrathin sectionswere stained with 5% methanolic uranyl acetate and Rey-nold's lead citrate and examined with a Zeiss EM-10 electronmicroscope at 80 kV.

RESULTSTicks from Connecticut contained rickettsialike microor-

ganisms in hemocytes that reacted with conjugated andunlabeled antisera to E. canis. From 1989 to 1991, 609 I.dammini ticks were examined for hemocytic microorgan-isms, and of these, 537 specimens were also tested for B.burgdorferi. Hemocytes from 59 (9.7%) nymphs, males, orfemales contained morphologically similar microorganismsthat reacted by direct or indirect FA staining to E. canisantisera (Table 1). The mean prevalence of infection rangedfrom 6.8 to 12.7% for males (n = 236) and females (n = 158),respectively. Rickettsialike organisms, having shared anti-gens with ehrlichiae, were observed in the hemocytes ofticks collected in the following towns of Connecticut: Bur-lington, Chester, Durham, Eastford, East Haddam, Lyme,

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2800 MAGNARELLI ET AL.

TABLE 2. Examination of nymphs and adults of I. dammini for rickettsialike microorganisms in hemocytes by directand indirect FA staining methods using antisera to E. canis

Direct FA Indirect FA (canine sera) Indirect FA (human sera)Sampling method

or source Total no. of No. (%) Total no. of No. (%) Total no. of No. (%)ticks tested positive' ticks tested positive ticks tested positive

Flagging 240 50 (20.8) 158 6 (3.8) 70 4 (5.7)Birds' 0 7 0 0Rodents' 0 5 ° ODeer or humans 129 7 (5.4) 0 0

Total 369 57 (15.5) 170 6 (3.5) 70 4 (5.7)

" Positive reactions indicate shared antigens with ehrlichiae.b Wood thrush (Hylocichla mustelina), ovenbird (Seiurus aurocapillus), and veery (Catharus fuscescens).Chipmunks and white-footed mice.

d Ticks found attached to hosts.

Old Lyme, and Wilton. Burlington and Eastford are innorthern Connecticut, while Wilton is in the southwesternpart of the state. The remaining towns are in south-central orsoutheastern Connecticut. In analyses for B. burgdorferi,146 (23.7%) of 617 I. dammini ticks harbored these spiro-chetes in their midguts. The prevalence of spirochetal infec-tion was more than twofold greater for adults (28.9%) thanthat of nymphs (13%). Despite relatively high infection ratesfor B. burgdorferi, the number of ticks simultaneouslycarrying this spirochete and hemocytic rickettsialike micro-organisms was low. Of the 537 nymphs and adults tested forboth bacteria, 36 (6.7%) specimens had coexisting organ-isms. Examination of 43 females and 30 males of D. variabi-lis, collected in East Haddam and Lyme, Connecticut,revealed hemocytic rickettsialike organisms in 1 female tickand 4 male ticks. No B. burgdorferi were detected.

Rickettsialike microorganisms were observed in the cyto-plasm of tick hemocytes by direct or indirect FA stainingprocedures. Although the percentage of positivity wasnearly threefold greater by direct FA staining methods(Table 2), intense fluorescence of microorganisms wasnoted, regardless of the assay method used (Fig. 1). Paralleltests of hemolymph preparations were conducted on 9female and 12 male I. dammini ticks. The hemocytes of threefemales and four males were positive by indirect FA stainingmethods using antiserum to E. canis or to a monoclonalantibody to Ehrlichia species (134-5B1). Hemocytes fromanother male tick reacted by indirect FA staining with thismonoclonal antibody and by direct FA staining. Hemocytes

FIG. 1. Rickettsialike organisms in the cytoplasm of a hemocytefrom a female I. dammini following direct FA staining with antibod-ies to E. canis. Bar = 5 ,um.

from the remaining 13 ticks were negative by these methods.Not all hemocytes of positive ticks were infected. Morulae,a growth stage described for ehrlichial infection (26, 36),were not observed. The number of positive I. damminiincluded questing ticks (i.e., ticks searching for vertebratehosts) as well as those that had been feeding on mammals.However, positivity for rickettsialike microorganisms wasnearly fourfold greater for the unfed, questing ticks.

In specificity studies, canine antiserum to E. canis did notreact with W. persica or with the SFG rickettsiae by indirectFA staining methods. Similar results were recorded whenhuman sera from spotted fever patients were tested againstE. canis antigen and when the direct FA reagent for E. caniswas screened against W. persica and the SFG rickettsiae.However, homologous reactions for E. canis, W. persica,and R. rickettsia were positive at titers of 1:5,120 or greater.Cross-reactivity was noted when conjugated or unlabeledantiserum to E. canis was tested against E. risticii antigen(titer = 1:1,280).

Parallel tests were conducted to determine whether humanserum containing antibodies to R. rickettsii (titer = 1:4,096)would react with the hemocytic rickettsialike microorgan-isms in female I. dammini. Of the 20 hemolymph prepara-tions positive by direct FA staining with antiserum to E.canis, none of the duplicate preparations from these ticksreacted when sera from spotted fever patients were tested byindirect FA staining.Numerous rickettsiae were observed by electron micros-

copy in ovarian tissue of adult female I. dammini (Fig. 2).The reactivity of these organisms to conjugated E. canisantiserum is unconfirmed. However, hemocytes from theseticks contained rickettsialike organisms that reacted posi-tively with FITC-labeled antibodies to E. canis. Ovariantissues were identified by the presence of intercellular cyto-plasmic bridges and extensive microtubules within the hostcell cytoplasm (Fig. 2B), as described previously by Brinton(6) and Raikhel (35). No rickettsiae were found by examiningserial sections of other internal organs, including the Mal-pighian tubules, midgut epithelium, nerves, salivary glands,and tracheae.By electron microscopy, the individual rickettsiae found

in tick ovaries were uniformly rod-shaped and measured 0.9to 1.5 ,um long and 0.4 to 0.5 ,um wide (Fig. 3A). Therickettsiae were surrounded by a prominent electron-lucenthalo zone and were delineated by two distinct unit mem-branes: an outer cell wall that was characteristically rippledand an inner cytoplasmic membrane, separated by an elec-tron-lucent periplasmic space (Fig. 3B). The various shapes

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FIG. 2. (A) Oocyte from an unfed female I. dammini showing numerous rickettsiae (R) within the cytoplasm. N, host cell nucleus. Bar= 2 ,um. (B) Longitudinal section of an intercellular bridge (IB) and associated microtubules (MT) within rickettsia-infected ovarian tissue.R, rickettsia. Bar = 0.5 ,um.

of the rickettsiae observed in ovarian cells were due to theorientation of microorganisms at the time they were sec-

tioned for electron microscopy. True pleomorphic formswere not observed. Also, electron-lucent halo zones some-

times overlapped and appeared deceptively like vacuoles butwere clearly without membranes.The cytoplasm was amorphous and composed of a dense

granular matrix. The cytoplasm was noticeably void of any

fibrillar reticulation, nuclear inclusion bodies, or well-definedvacuoles but often contained a single small (65- to 120-nmdiameter) crystalline inclusion body that possessed a distinctlattice structure (Fig. 3C).

Rickettsiae were confined to the host cell cytoplasm, andreproduction appeared to occur by binary fission (Fig. 3D).A variable number of lysosomelike organelles that containedsmall vessicles surrounded by concentric membranes werealso observed (Fig. 3E). These were randomly dispersedthroughout the cytoplasm and appeared to engulf individualrickettsiae.

DISCUSSION

Rickettsialike microorganisms are present in the hemo-cytes of I. dammini and D. variabilis. However, it is unclearwhether or not these organisms, which apparently shareantigens with ehrlichiae, are pathogenic to humans or do-mesticated animals. They could be avirulent. Initial attemptsto isolate these organisms from I. dammini in cell culturehave been unsuccessful thus far. Ticks are suspected vectorsof ehrlichiae, but conclusive experimental evidence linkingthe pathogen, vector, and vertebrate host for Potomac horsefever and human ehrlichiosis in the United States is lacking.

Isolation of etiologic agents and subsequent studies withticks and laboratory animals are required to demonstratetransmission and pathogenicity. Such experiments havebeen performed for E. canis, R. sanguineus, and dogs (20)and can be performed for other ehrlichial agents as well.However, brown dog ticks, unlike I. dammini, are relativelyhost specific (28) and prefer dogs over other mammals. If theetiologic agent of human ehrlichiosis is E. canis, then it islikely that additional tick vectors are involved.Canine ehrlichiosis occurs in Connecticut (25) and several

other states where there are multiple tick species. Asidefrom dogs and other wild canids, such as coyotes and redand gray foxes, that are reservoirs for E. canis, it is unknownif other wildlife (e.g., rodents, lagomorphs, or deer) are

likely reservoirs for this organism or other ehrlichiae. InEurope, I. ricinus is a vector of Ehrlichia phagocytophila,the etiologic agent of tick-borne fever in sheep and cattle(37). Deer are believed to be natural hosts for this granulo-cytic ehrlichia. Thus, alternative reservoir hosts for E. canisand E. risticii should be considered.The number of I. dammini harboring hemocytic rickettsi-

alike microorganisms was larger than the number of D.variabilis adults infected with SFG rickettsiae (24) but was

smaller than the number infected with B. burgdorferi (21).Although ticks do not efficiently pass ehrlichiae or B. burg-dorferi transovarially (22, 26), transstadial transmission ofthese bacteria does occur (3, 26). Therefore, as in Lymeborreliosis, the competence of vertebrate reservoirs forehrlichiosis is probably an important epidemiological factorand warrants further study.

In tests of the specificity of conjugated or unlabeledantisera to E. canis, there was no cross-reactivity with W.

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FIG. 3. (A) Electron micrograph of a rickettsia from I. dammini. Note the distinct halo zone (HZ) surrounding the organism. Bar = 0.1,um. (B) Higher magnification of the rickettsia, showing the organization of the cell envelope. CM, inner cytoplasmic membrane; CW, outercell wall; HZ, halo zone; PS, periplasmic space. Bar = 0.1 ,um. (C) Higher magnification of the same rickettsia, showing crystalline inclusionbody (arrow) within the cytoplasm of the rickettsia. Bar = 0.05 ,um. (D) Rickettsia undergoing binary fission. Arrows denote point ofconstriction. Bar = 0.1 ,um. (E) Lysosomelike organelles (L) found within rickettsia-infected ovarian tissue. Note apparent degradation ofrickettsiae (R). Bar = 0.5 ,um.

persica or SFG rickettsiae. Failure to cross-react with SFGor typhus-group rickettsiae agrees with earlier reports (26,36, 38). Similarly, human sera containing antibodies to E.canis or a related agent were negative in tests with thefollowing antigens: R. rickettsii, Rickettsia typhi, Coxiella

burnetii, Francisella tularensis, and B. burgdorferi (14).However, serologic cross-reactivity among ehrlichiae, suchas E. sennetsu, E. canis, E. equi, and E. risticii, has beenreported previously (14, 26, 36). Although not known tooccur in the United States, rickettsiae in the genus Cowdria,

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RICKETTSIAE AND B. BURGDORFERI IN TICKS 2803

within the tribe Ehrlichieae, share common antigens withmembers of the genus Ehrlichia, particularly E. equi (19).Thus, a variable degree of serologic cross-reactivity amongehrlichiae is probable. SFG rickettsiae also infect tick hemo-cytes (8, 23, 24), but in Connecticut, the prevalence ofinfection in D. variabilis and I. dammini was usually lessthan 3% (23, 24). Although the antisera used in the presentstudy were produced against E. canis, the hemocytic rick-ettsialike organisms observed in I. dammini and D. variabilismay not be this bacterium but another closely relatedorganism. Elevated positivity rates recorded by direct FAstaining were probably due to higher concentrations of IgGantibodies and overall better reagent quality.

Ticks can harbor symbiotes, such as W. persica, in ovarianand Malpighian tubules (16). However, these rickettsiae donot commonly occur in tick hemocytes. Moreover, on thebasis of analyses of 16S rRNA sequences, W. persica appearsto be more distantly related to the ehrlichiae than are the SFGand typhus-group rickettsiae (45). Representatives ofthe genusRickettsia (tribe Rickettsieae) were placed along with E. risti-cii (tribe Ehrlichieae) in the alpha subdivision of the purpleeubacteria (class Proteobacteria), whereas W. persica (tribeWolbachieae) was assigned to the gamma subdivision ofthesebacteria. It is unlikely that the hemocytic microorganismsobserved in the present study are in the genus Wolbachia.Although members of the genera Rickettsia and Ehrlichia aremore closely related phylogenetically, there is minimal or noserologic cross-reactivity between these genera.The rickettsiae observed in ovarian tissues of I. dammini do

not appear to be morphologically similar to ehrlichiae. Theehrlichiae are round to ovoid, have an inner structure ofvariable electron density, and are contained within character-istic membrane-lined vacuoles called morulae (18, 36, 37, 42).In contrast, the rickettsia found in the ovaries of I. damminihas morphological characteristics that are similar to those ofthe SFG rickettsiae (47) (i.e., size, shape, nature of cell walland cytoplasmic membrane), including an apparently non-pathogenic isolate from A. americanum called WB-8-2 (11).Ultrastructurally, these rickettsiae and those observed in theovaries of I. dammini have a diplo-elliptical shape and densegranular cytoplasm that contains crystalline inclusions. AI-though the nature and function of these crystalline inclusionsare unknown, they also have been observed in Rickettsiacanada (7) and infrequently in degenerating R. rhipicephali(15).Aside from the slightly larger diameter of WB-8-2 (0.68

versus 0.4 to 0.5 ,um), a major difference between theserickettsiae and the microorganisms observed in the ovariesof I. dammini appears to be the sites of infection within thetick. Most abundant in the ovaries and Malpighian tubules ofA. americanum (11), WB-8-2 usually invades other tick hosttissues. The rickettsialike microorganisms in I. dammini,however, are apparently limited to hemocytes and ovariantissue. We were unable to specifically test rickettsialikeorganisms in I. dammini against antisera to WB-8-2, becausehomologous antiserum to this rickettsia was unavailable.The hemocytic organisms in I. dammini did not cross-react

when human sera from spotted fever patients were tested.Additional serologic test results revealed no cross-reactivitybetween W. persica antisera and the hemocytic rickettsialikeorganisms in I. dammini. The absence of pleomorphic forms,nuclear inclusion bodies, and fibrillar reticulation in the cyto-plasm of ovarian rickettsiae also clearly distinguishes themfrom symbiotic Wolbachia spp. (16, 46).

Because of the difficulty in isolating tick hemocytes forelectron microscopy, ultrastructural studies of the microor-

ganisms in these cells have not been conducted. Likewise,ovarian rickettsiae in I. dammini have not been testedserologically because of nonspecific autofluorescence prob-lems. However, proper characterization of these rickettsiaecan be accomplished after the organisms have been isolatedand cultured. Therefore, we cannot exclude the possibilitythat there are two or more distinct rickettsiae in these ticks.Nonetheless, our documentation is the first report of rick-ettsiae in ovarian tissues of I. dammini.Hemocytic rickettsialike organisms coexisted with B.

burgdorferi in I. dammini. Simultaneous transmission of B.burgdorferi and Babesia microti, the etiologic agent ofhuman babesiosis, has been suggested for I. dammini (34),but it is unclear whether single infections of these agents aremore prevalent than dual infections are. Little is knownabout the compatibility of multiple pathogens in ticks, phys-iological factors that may inhibit or favor coexistence, andtransmission efficiency. Regardless, larvae, nymphs, andadults of I. dammini have a broad host range, and withincreased tick abundance and contact with wildlife, there ispotential for acquiring and transmitting a variety of micro-organisms to human beings and domesticated animals.

Unexplained febrile illnesses occur in persons living intick-infested areas (14). There is greater public awareness ofticks and Lyme borreliosis, but clinical diagnosis of thisdisease can be difficult when erythema migrans is atypical,absent, or not observed. Moreover, the nonspecific clinicalmanifestations of human ehrlichiosis, such as fever, arthral-gia, headache, and myalgia (36), also occur in B. burgdorferiinfections. The prevalence of human ehrlichiosis in foci forLyme disease is unknown. Further efforts should be placedon identifying rickettsiae and other microorganisms that maybe circulating in tick populations and on determining theirepidemiologic importance. In recent laboratory studies (4) ofa granulocytic ehrlichial organism, dogs fed upon by infectedlone star ticks showed serologic or clinical evidence ofehrlichiosis. Therefore, A. americanum should be includedalong with I. dammini and D. variabilis in investigations ofvector competency.

ACKNOWLEDGMENTS

We thank Frank Campbell, Merrily Gere, Cynthia Phillips,Aniello Infante, and Virginia Bladen for collecting ticks and fortechnical assistance. Robert Swihart of the Connecticut AgriculturalExperiment Station submitted additional ticks. Alan Barbour of theUniversity of Texas (San Antonio) provided murine monoclonalantibodies (H5332) to B. burgdorferi, and Gregory Dasch of theNaval Medical Research Institute (Bethesda, Md.) supplied mono-clonal antibodies to and antigens of W. persica and E. risticii. Weare also grateful to Joseph E. McDade of the Centers for DiseaseControl (Atlanta, Ga.) and Gregory Dasch for helpful comments on

an earlier version of this report.

REFERENCES1. Anderson, J. F. 1989. Epizootiology of Borrelia in Ixodes tick

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