JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/99/$04.0010

Aug. 1999, p. 2518–2524 Vol. 37, No. 8

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Invasion and Intracellular Development of the Human GranulocyticEhrlichiosis Agent in Tick Cell Culture

ULRIKE G. MUNDERLOH,1* STEVEN D. JAURON,1 VOLKER FINGERLE,2 LORENZ LEITRITZ,2

S. FRED HAYES,3 JOAN M. HAUTMAN,1 CURTIS M. NELSON,4 BRENT W. HUBERTY,4

TIMOTHY J. KURTTI,1 GILBERT G. AHLSTRAND,5 BARBARA GREIG,6

MARTHA A. MELLENCAMP,7 AND JESSE L. GOODMAN4

Departments of Entomology,1 Plant Pathology,5 and Clinical and Population Sciences7 and Veterinary DiagnosticLaboratories,6 University of Minnesota, St. Paul, Minnesota, Rocky Mountain Laboratory, National Institutes

of Health, Hamilton, Montana,3 and Division of Infectious Diseases, Department of Medicine,4

and Max von Pettenkofer Institut,2 Universitat Munchen, Munich, Germany

Received 19 January 1999/Returned for modification 7 April 1999/Accepted 29 April 1999

Human granulocytotropic ehrlichias are tick-borne bacterial pathogens that cause an acute, life-threateningillness, human granulocytic ehrlichiosis (HGE). Ehrlichias within neutrophil granulocytes that invade tick bitesites are likely ingested by the vector, to be transmitted to another mammalian host during the tick’s next bloodmeal. Thus, the cycle of replication and development in the vector is prerequisite to mammalian infection, andyet these events have not been described. We report tick cell culture isolation of two strains of the HGE agentdirectly from an infected horse and a dog and have also established a human isolate from HL60 culture in tickcells, proving that the blood stages of the HGE agent are infectious for tick cells, as are those replicating inthe human cell line HL60. This required changes to the culture system, including a new tick cell line. In tickcell layers, the HGE agent induced foci of infection that caused necrotic plaques and eventual destruction ofthe culture. Using the human isolate and electron microscopy, we monitored adhesion, internalization, andreplication in vector tick cells. Both electron-lucent and -dense forms adhered to and entered cells by amechanism reminiscent of phagocytosis. Ehrlichial cell division was initiated soon after, resulting in endo-somes filled with numerous ehrlichias. During early development, pale ehrlichias with a tight cell walldominated, but by day 2, individual bacteria condensed into dark forms with a rippled membrane. These maybecome compacted into clumps where individual organisms are barely discernible. Whether these are part ofan ehrlichia life cycle or are degenerating is unknown.

Human granulocytic ehrlichiosis (HGE) agents are obligateintracellular prokaryotes that are transmitted to mammalianhosts via the bite of an infected tick. They cause an emergingdisease, HGE (1, 9), that is characterized by an acute, some-times fatal febrile syndrome most commonly accompanied bymalaise, headache, myalgia, and arthralgia. Laboratory find-ings include leukopenia, thrombocytopenia, anemia, and ele-vated serum transaminases (1, 17). Stained blood films revealthe presence of groups of organisms, called morulae, enclosedin a common vacuole within neutrophil granulocytes. Besideshumans, horses and dogs also appear to be susceptible to theHGE agent (2, 13, 14, 18, 20), and white-footed mice (8, 30, 40)and possibly white-tailed deer (4, 24) may act as a reservoir innature. In North America, the HGE agent is transmitted by theticks Ixodes scapularis (12, 31, 40) and Ixodes pacificus (34, 36),and in Europe, the closely related tick Ixodes ricinus appears tobe the main vector (16, 32). Serologic studies place the HGEagent within the same geographic range as the Lyme diseasespirochete, Borrelia burgdorferi, which shares the same vectortick (18, 37).

Demonstration of the agent in its tick vector has beenachieved by PCR amplification of HGE agent-specific nucleicacid sequences based on 16S ribosomal DNA (rDNA) (7, 10,31) and visualization by light microscopy in salivary glandsstained with the Feulgen technique (40). It appears that levels

of infection in ticks are either extremely low or that, in thevector, the HGE agent exists in a form that is not easily rec-ognizable by cytologic or serologic methods. We previouslyreported culture isolation of a closely related tick-borne patho-gen of cattle, Anaplasma marginale, and the MRK strain ofEhrlichia equi in cell line IDE8 (27, 29). Here, we describedirect cultivation from blood of two new primary isolates of theHGE agent from a horse and a dog and describe invasion oftick cells in culture by a human patient isolate, as well as itsintracellular morphogenesis. To achieve this required changesto the culture system, including the use of a new cell line.

MATERIALS AND METHODS

Tick cell culture. Cell line ISE6 (26) from the tick I. scapularis was used. Stockcultures were maintained as described elsewhere (28), except that they weregrown at 34°C. Also, the L-15B culture medium was modified by addition ofone-fourth water by volume, to lower the osmotic pressure from approximately420 mosM/liter to approximately 315 mosM/liter (L-15B300). Fetal bovine serum(5%; Sigma, St. Louis, Mo.), tryptose phosphate broth (10%; Difco, Detroit,Mich.), and lipoprotein concentrate (0.1%; ICN, Irvine, Calif.) were added, andthe pH was adjusted to 7.0 to 7.2. Medium was changed once a week.

Ehrlichia culture. Primary isolates from a dog and a horse were obtained fromanimals naturally infected in Minnesota. Blood from the horse was brought tothe laboratory the same day as collected, and dog blood was taken from a patientpresenting to the small animal clinic in St. Paul, Minn. Blood was drawn intoEDTA as anticoagulant. Buffy coat cells were harvested after centrifugation for10 min at 500 3 g at room temperature and washed once in L-15B300. Buffy coatand contaminating erythrocytes were layered onto a 25-cm2 (Sarstedt, Newton,N.C.; vented plug cap) culture of ISE6 cells with 5 ml of L-15B300 supplementedas for routine cell stock maintenance, plus 0.25% NaHCO3 and 25 mM HEPES,pH 7.5, referred to as ehrlichia medium. Cultures were incubated in a candle jarat 34°C as described elsewhere (29), and medium was changed twice weekly,taking care not to remove the erythrocytes. If erythrocytes became depleted due

* Corresponding author. Mailing address: Department of Entomol-ogy, University of Minnesota, 219 Hodson Hall, 1980 Folwell Ave., St.Paul, MN 55108. Phone: (612) 624-3688. Fax: (612) 625-5299. E-mail:munde001@maroon.tc.umn.edu.

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to lysis during the first 1 to 2 months, fresh, washed human erythrocytes wereadded (approximately 108 per 5 ml of culture).

To transfer a human patient-derived HGE isolate from HL60 to tick cellculture, HL60 cells infected 30 to 90% with isolate HGE-MN1 (17) in the fifthpassage in vitro were added to ISE6 cells at a ratio of 1:20. Washed humanerythrocytes were added, and cultures were incubated as described above.

Once the ehrlichias from animal blood or HL60 cultures became establishedin ISE6 cells, usually by the third to fourth passage, cultures were incubated intightly capped flasks (i.e., without CO2). During the early passages, 1/5 to 1/10 ofan infected cell layer was transferred to a new culture of ISE6 cells. Later, theinoculum for pathogen maintenance was reduced to 1 to 5%, and transfers weremade once every 7 to 10 days. Addition of fresh tick cells during that time wasnot needed. Progress of infection in ISE6 cells was monitored by phase-contrastmicroscopy or by examination of Giemsa-stained cell spreads.

To test animal infectivity of HGE isolates grown in ISE6 cells, cultures inwhich at least 70% of the cells were infected were resuspended in their medium,and 0.5 ml of the suspension was inoculated intraperitoneally into 3-week-oldhamsters (Mesocricetus auratus) or 4-week-old mice (C3/HeJ). From day 7 post-inoculation (p.i.), tail blood smears were prepared and stained with Giemsa’sstain to detect infected neutrophil granulocytes by light microscopy. Animalswere killed by exposure to CO2 gas at 2 weeks p.i. and exsanguinated by cardiacpuncture. DNA from EDTA-blood was purified with the PureGene reagent kit(Gentra Systems, Inc., Minneapolis, Minn.). Animals were cared for and used inaccordance with the rules and regulations established by the Institutional AnimalCare and Use Committee of the University of Minnesota.

PCR and sequencing. To verify the identity of the new equine and canineisolates, DNA was purified from infected ISE6 cultures with either ISOQuick(Orca Industries, Bothell, Wash.) or PureGene (Gentra). Ehrlichia DNA wasamplified with the genus-specific primers PER1 and -2 and a species-specificprimer pair, GER3 and -4, and electrophoresed, stained, and visualized asdescribed elsewhere (17). For comparison, DNA extracted from HL60 cellsinfected with HGE-MN1 and -2 (17), IDE8 cells infected with Ehrlichia canis(15), DH82 cells infected with Ehrlichia chaffeensis (courtesy of J. E. Dawson,Centers for Disease Control and Prevention, Atlanta, Ga. [11]), and uninfectedISE6 cells were also subjected to PCR under the same conditions.

16S rRNA gene sequence analysis. 16S rDNA to be sequenced was PCRamplified with primers PER3 (59 ATG CAT TAC TCA CCC CTC TG 39) andGER4 (59 AAG TGC CCG GCT TAA CCC GCT GGC 39), which span bases 1to 20 and 1077 to 1101, respectively (17), of the reported sequence for the agentof HGE (GenBank accession no. U02521). Both strands of the amplificationproduct were sequenced with the oligonucleotide primers PER1 (bp 187 to 211),PER2 (bp 616 to 638), PER3 (bp 1 to 20), PER4 (bp 92 to 111), PER5 (bp 818to 837), PER6 (bp 925 to 943), GER3 (bp 950 to 973), and GER4 (bp 1077 to1101) (17). Sequencing was carried out in a 377XL DNA sequencer (AppliedBiosystems GmbH, Darmstadt, Germany) by the Taq DyeDideoxy terminatormethod. Sequence analyses were done with DNAMAN for Windows 95 (LynnonBioSoft, Quebec, Quebec, Canada). Sequence data for comparison were ob-tained from GenBank.

Time course of infection. HGE-MN1-infected ISE6 cultures (90 to 100% ofcells infected, ,10 passages in ISE6 tick cells) were resuspended and passed fivetimes through a 25-gauge needle by means of a 5-ml LuerLock syringe to disruptthe cells and liberate the ehrlichias. After low-speed centrifugation of the result-ing suspension to remove large debris and intact cells (175 3 g, 10 min at roomtemperature), ehrlichias in the supernatant were sedimented for 15 min at13,000 3 g. Pellets were resuspended in ehrlichia medium, mixed with approx-imately 2.5 3 107 ISE6 cells at a ratio of one infected to one uninfected cell, andseeded into a tissue culture plate (24 well; Nunc, Roskilde, Denmark), 0.5 ml perwell. The plate was incubated at 34°C in a candle jar. After 30 min, the plate wasgently rocked, the supernatant was discarded, and cell layers were rinsed oncewith unsupplemented L-15B300 to remove nonadherent ehrlichias. Wells wererefilled with 0.5 ml of ehrlichia medium. At 1, 2, 4, 8, 12, 24, 48, and 96 h p.i., cellsfrom two wells were resuspended and pipetted into 1 ml of fixative each (seebelow). The suspension was centrifuged for 5 min at 175 3 g, and the supernatantwas replaced with fresh fixative. Cells from a third well were spun onto micro-scope slides, fixed in absolute methanol, and stained with Giemsa’s stain. Theexperiment was repeated twice, with ehrlichias of the same isolate and at equiv-alent passage levels.

Electron microscopy. Cultures were processed for electron microscopy asoutlined elsewhere for E. equi (29). Briefly, cells were fixed in modified Ito’sfixative (19, 22) and treated with 0.1% tannic acid in water to enhance demon-stration of ehrlichial membranes. Postfixation was done in 0.5% osmium tetrox-ide with 0.8% potassium-ferrocyanide as a reducing agent. Cells were soaked in0.1% uranyl acetate overnight, dehydrated in ascending concentrations of etha-nol, and embedded in Spurr’s low-viscosity resin (EM Sciences, Cherry Hill,N.J.). Thin sections were stained with lead citrate (35, 38) and examined on aPhilips EM12 or a Hitachi HU-11E-1 electron microscope.

RESULTS

Primary isolation and transfer from HL60 cells. The behav-ior of ehrlichias from infected animal blood and that of ehr-

lichias from HL60 cells were similar and are described togetherhere. Successful isolation of the HGE agent, whether frominfected blood or from HL60 culture, required alteration of theculture conditions and the use of a different tick cell line.Initially, we used the cell line (IDE8) and culture conditions(i.e., undiluted L-15B, no blood supplementation) that led tothe successful isolation of the MRK strain of E. equi. Everytime, infected tick cells were seen in Giemsa-stained smears by1 week p.i., but by 3 weeks, infected cells could no longer bedemonstrated. Despite repeated attempts, no isolates wereobtained in IDE8 cells or when undiluted L-15B was used. Wethen replaced cell line IDE8 with cell line ISE6, lowered themedium osmolarity, and included erythrocytes in the cultures.Under those circumstances, the ehrlichias successfully becameestablished in tick cell culture, continued to multiply, and ef-ficiently spread through the cultures, eventually infecting 90%or more of the cells. During the first 4 to 8 weeks p.i., ehrlichialgrowth was erratic. Addition of erythrocytes was beneficial andstimulated development of ehrlichias. Once regular subcul-tures could be made, an atmosphere reduced in O2 and en-riched in CO2 was no longer required, cultures could be main-tained outside the candle jar, and blood supplementation wasunnecessary. HGE isolates propagated in tick cell culture re-tained infectivity for hamsters or mice until at least the 28thpassage in tick cells and caused infection of peripheral bloodneutrophil granulocytes as demonstrated in Giemsa-stainedblood smears and by PCR. In ISE6 cells, the appearance of theehrlichial inclusions was similar to that of the E. equi inclusionsin IDE8 cells. Notably, single colonies of ehrlichias were muchlarger than morulae in granulocytes or HL60 cells, and patho-gen morphology was more variable, ranging from masses ofhighly pleomorphic, bluish-staining organisms to tiny coccoid,magenta forms and deep inky blue compacted bodies probablyrepresenting the dense colonies described below.

With regular subculturing and at higher dilutions of theinoculum (1:50 to 1:100), distinct foci of ehrlichial multiplica-tion were demonstrable in the cell layer by phase-contrastmicroscopy. Single infected cells (Fig. 1A) lysed, releasing theorganisms, which in turn infected other neighboring cells, en-larging the plaque (Fig. 1B). Dead cells in the center detachedand disintegrated, causing the appearance of holes in the celllayer (Fig. 1C). Such cytopathic changes were not seen inuninfected cultures.

PCR and nucleotide sequence analysis. Primers PER1 and-2 mediated amplification of DNA of the appropriate size fromall ehrlichia sources but not from tick host cell DNA (data notshown), while GER3 and -4 produced a 150-bp fragment in thePCR only when template DNA was from granulocytic ehrlich-ia-infected cultures (i.e., isolates HGE-MN1 and -2, as well asthe canine and the equine HGE isolates [Fig. 2]). This estab-lished and confirmed that all three strains growing in ISE6 tickcells were HGE isolates. Analysis of a 1,098-bp stretch (bp 1 to1098) of 16S rDNA additionally revealed 100% identity of thetwo new animal isolates with the first described isolate (17).

Invasion and morphogenesis in ISE6 cells. For each timepoint p.i., several sections from the two experiments were ex-amined to ensure that the phenomena observed were repre-sentative of ehrlichial behavior. Within 1 h p.i., ehrlichias werefound attached to tick cells (Fig. 3A and B). Attached andinvading organisms were condensed to a varying degree asindicated by a loose, rippled membrane and included verydense, dark forms (Fig. 3A) as well as paler, mottled organ-isms. By 2 h p.i., spreading of the ehrlichial membrane at thetick cell attachment site was noticeable (Fig. 3B, arrows), andat 4 h p.i., host cells had become actively engaged in taking upehrlichias by a process that resembled phagocytosis (Fig. 3C).

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Organisms at the cell surface caused protrusion of a cytoplas-mic lip that eventually enclosed and internalized the bacterium(Fig. 3D). There was indication that focal points of adhesionbetween the cell and ehrlichial membrane (Fig. 3C, arrow andarrowheads) migrated and shifted position around the bacte-rial body until the lips of the phagocytic pit met, enclosing it.These points of attachment appeared preserved within theearly endosome (Fig. 3D, arrows), suggesting a receptor-me-diated interaction. Between 4 and 8 h p.i., ehrlichias that wereinitially resting singly in the endosome (Fig. 4A) were observedto show initial evidence of cell division (Fig. 4B), leading to theappearance of small morulae comprising just a few organismsas early as 12 h p.i. (Fig. 4C). Thereafter, ehrlichial cell divisionand development proceeded rapidly, and by 48 h p.i. distendedendosomes harbored large numbers of bacteria, including thefirst condensed, electron-dense forms (Fig. 4D). After thattime, development became asynchronous, as ehrlichias liber-ated from tick host cells reinvaded and initiated new infections.An examination of ehrlichias near the endosomal peripheryshowed close apposition of bacterial and host endosomal mem-branes (Fig. 5A and B, arrowheads), possibly indicative ofparasite-host metabolic interaction. In older (96 h p.i.) cul-tures, ehrlichial colonies comprising only pale, electron-lucentforms coexisted with those harboring appreciable numbers($50% of bacteria in the section) of condensing organisms(Fig. 5C). In some cells, these became compacted into massesof bacteria in which individual organisms could just barely bedelineated by their membranes (Fig. 5D).

DISCUSSION

In recent years, a number of previously unknown pathogenshave been linked to emergence of new human disease entities.It is noteworthy that several of these are arthropod-borne, inparticular tick-borne; have a feral animal reservoir; or areclosely related to animal pathogens. Ticks that feed on a widerange of hosts may be vehicles for the transfer of pathogens tonew host species (41), aiding the emergence of new diseases.One such group of emerging pathogens are the human ehrli-chiosis agents, E. chaffeensis (11) and the recently discoveredHGE agent (1, 9, 17). This is the newest member of the Ehr-lichia phagocytophila group that also includes pathogens ofruminants, horses (E. equi) (25), and dogs (18) and appears tohave acquired the ability to infect humans. Pathogens in that

group are so closely related that they cannot be distinguishedby 16S rDNA nucleotide sequence analysis (12, 16) and couldconceivably be considered strains of one species. Nevertheless,the behavior of the HGE agent compared with that of E. equiin tick cell culture indicated that biological differences existbetween the two and might be responsible for their seeminglydivergent host spectrum in nature. For example, despite re-peated attempts, we have not been able to transfer the HGE-MN1 isolate from ISE6 to IDE8 cells, and while the HGEisolates are infectious for small laboratory rodents, E. equi isnot (data not shown).

Events which take place in the tick are of importance withrespect to their transmission to mammals and contribution toor influence on pathogenicity. Unfortunately, the portion ofthe life cycle which the HGE agent spends inside the tick is an

FIG. 1. Phase-contrast microscopy of plaque formation by the HGE agent in cultured tick cells. (A) Single infected cell (arrow). Note distention of the cellmembrane and granular cell contents representing ehrlichias. Bar, 40 mm. (B) Small infected focus of highly granular cells (arrows). Bar, 40 mm. (C) Plaque that hasformed by lysis and detachment of infected cells (arrows). Bar, 100 mm.

FIG. 2. Agarose gel electrophoresis of PCR products amplified with primersGER1 and GER3, specific for 16S rDNA of ehrlichias in the E. phagocytophilagroup. Shown is a 1.75% agarose gel stained with ethidium bromide. TargetDNA was purified from different materials as follows: lanes HGE1 and HGE2,I. scapularis tick cell culture, line ISE6, infected with the human isolate HGE-MN1 and HGE-MN2 (17), respectively; lane E. chaff, DH82 cells infected withE. chaffeensis (11); lane E. canis, I. scapularis tick cell cultures, line IDE8, infectedwith E. canis (15); lane Canine, I. scapularis tick cell culture, line ISE6, infectedwith the canine HGE isolate; lane Equine, I. scapularis tick cell culture, lineISE6, infected with the equine HGE isolate; lane ISE6 Cells, I. scapularis tick cellcultures, line ISE6, uninfected host cell control; lane H2O, no target control.

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enigma and has barely been investigated. A single publicationshows the ultrastructure of E. canis in the tick (39). It clearlydemonstrates morphological differences between the bloodand the tick stages of this species, including condensation ofehrlichias residing in the salivary glands. It is reasonable toassume that the granulocytotropic ehrlichias, too, differ in theirappearance in the tick versus in the mammal. E. canis in tickcell culture resembles the forms found in the tick (15), and theHGE agent in I. scapularis cells might be representative of itsarthropod phase. In any case, the appearance of the HGEagent in tick cells is very different from that in mammaliancells, the neutrophil granulocyte as well as HL60 cells, hintingat specific adaptations to these divergent hosts. Moreover, andas previously shown with E. equi in the I. scapularis cell line

IDE8 (29), granulocytotropic ehrlichias in ISE6 cells display astriking array of morphologic forms. Many host cells becomecompletely filled with ehrlichias and are enlarged to severaltimes their original size. In addition, ISE6 cells are cultured atmore than 10 times the density of HL60 cells, i.e., 2 3 106 to3 3 106/ml for ISE6 versus 1 3 105/ml for HL60. In conjunc-tion with the higher pathogen load per cell, this results in alarger yield of antigen per milliliter of culture in a mediumsupplemented with half the amount of fetal bovine serum usedfor HL60.

As a first step in the investigation of the cellular interac-tion between pathogen and vector cell, we have analyzed theagent’s morphogenesis in this culture system under environ-mental conditions of temperature (34°C) and gaseous atmo-

FIG. 3. Sequence of adhesion to and invasion of cultured vector tick cells by the HGE agent. (A) One hour p.i.; condensed ehrlichia with rippled membrane,attached to a tick cell. Note intimate contact with the tick host cell membrane as indicated by the arrows. (B) Two hours p.i.; the membrane of an attached ehrlichiahas spread out along the tick host cell membrane (arrows). (C) Four hours p.i.; uptake of ehrlichia into host cells by a process resembling phagocytosis. Note protrusionof a cytoplasmic lip in preparation of internalization of the ehrlichia. Arrow and arrowheads indicate focal points of adhesion between the cell and ehrlichial membrane.(D) Completely internalized ehrlichia. Arrows indicate points of initial attachment that were apparently preserved within the early endosome. Bars, 0.2 mm.

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sphere (low O2 and high CO2) that mimic the tick’s internalenvironment during a blood meal (23). Presumably, infectionof the arthropod vector results when the tick ingests ehrlich-ia-infected neutrophil granulocytes that enter the tick bite site.Peripheral blood neutrophils most likely were also the sourceof ehrlichias that invaded the tick cells in vitro. Our ability totransfer HGE isolates from the human cell line HL60 to tickvector cells further suggests that these cultured organisms areequivalent, at least in this aspect, to the granulocyte forms.Both primary isolates, and the human isolate which had beenpassaged in HL60 cells six times, noticeably benefited from theaddition of erythrocytes to the culture. Iron presented in abiologically active form, e.g., bound to transferrin, has beenshown to be utilized by E. chaffeensis (3) and generally plays arole as a virulence factor in pathogenic bacteria.

Some aspects of ehrlichial entry into ISE6 tick cells ap-

peared similar to that described for A. marginale in IDE8 tickcell culture (5), but there were also differences including amuch higher degree of pleomorphism in the ehrlichias as wellas the fact that they did not grow in IDE8 cells. Although thetwo pathogens are closely related by 16S rDNA analysis (6),they differ markedly in their host cell tropism in the mammal(the erythrocyte is the only known target cell of A. marginale)and behavior in the vector. Such traits are likely the product ofgenes that evolve rapidly to allow divergence into new ecolog-ical niches and may not be reflected in the much more stableribosomal genes.

Unlike the granulocytotropic ehrlichias, A. marginale oftencauses heavy infections in the tick. This facilitated light andelectron microscopic analyses of its development in that host(21) and demonstrated that morphogenesis in tick cell cultureis comparable to the situation in vivo (5, 27). Therefore, tick

FIG. 4. Replication of the HGE agent in tick cells in vitro. (A) Single ehrlichia enclosed with endosome, 4 h p.i. Bar, 0.4 mm. (B) Ehrlichia with enfolding membrane(arrows), possibly the first evidence of division by 8 h p.i. Bar, 0.4 mm. (C) At 12 h p.i., replication has been initiated, leading to appearance of small morulae (arrows).Bar, 1 mm. (D) By 48 h p.i., distended endosomes harbor large numbers of bacteria, including the first electron-dense forms (arrow). Bar, 2 mm.

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FIG. 5. Late developmental forms of the HGE agent in tick cell culture. (A) From 72 h p.i., tick cells contained large endosomes more or less densely filled withhighly pleomorphic ehrlichias. Bar, 1 mm. (B) Inset from panel A, enlarged. Ehrlichias near the endosomal periphery showed close contact between bacterial and hostendosomal membranes (arrows). Bar, 0.2 mm. (C) In older (96 h p.i.) cultures, darker-staining, condensing organisms (arrows) coexisted with pale forms inside ehrlichialcolonies. Bar, 1 mm. (D) In some cells, these colonies became compacted into masses of bacteria in which individual organisms could barely be delineated by theirmembranes. Bar, 0.2 mm.

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cell culture may also be a valid in vitro model for the tick phaseof the ehrlichial life cycle. While there were similarities in thein vitro development of the two pathogens, the differencesincluded a much higher degree of pleomorphism in the ehr-lichias as well as the fact that they did not grow in IDE8 cells.Various morphological forms of both A. marginale and theHGE agent adhered to and were taken into the cells. Theseappeared more electron dense than the actively dividing intra-cellular organisms seen at 24 to 48 h p.i. and might be under-going development toward the electron-dense forms presentlater. Highly pleomorphic, densely compacted E. chaffeensis inmammalian cell culture has been described elsewhere as ab-errant and degenerating (33). However, in tick cells, ehrlichiaswere never found in association with debris typically present inlysosomes, such as fragments of cellular organelles or mem-brane whorls, suggesting that they were not being degraded.

ACKNOWLEDGMENTS

This study was supported in part by the following sources: grantsfrom the National Institutes of Health, no. 1R29AI42792 (to U.G.M.)and no. 1RO1AI40952 (to J.L.G.), and the University of MinnesotaExperiment Station (to T.J.K.).

We are grateful to Ann T. Palmer (Department of Entomology,University of Minnesota) for expert help in preparing the electronmicrographs.

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