Expression of multiple outer membrane protein sequence variants from a single genomic locus of Anaplasma phagocytophilum.

Anaplasma phagocytophilum is the causative agent of an emerging tick-borne zoonosis in the United States and Europe. The organism causes a febrile illness accompanied by other nonspecific symptoms and can be fatal, especially if treatment is delayed. Persistence of A. phagocytophilum within mammalian reservoir hosts is important for ensuring continued disease transmission. In the related organism Anaplasma marginale, persistence is associated with antigenic variation of the immunoprotective outer membrane protein MSP2. Extensive diversity of MSP2 is achieved by combinatorial gene conversion of a genomic expression site by truncated pseudogenes. The major outer membrane protein of A. phagocytophilum, MSP2(P44), is homologous to MSP2 of A. marginale, has a similar organization of conserved and variable regions, and is also encoded by a multigene family containing some truncated gene copies. This suggests that the two organisms could use similar mechanisms to generate diversity in outer membrane proteins from their small genomes. We define here a genomic expression site for MSP2(P44) in A. phagocytophilum. As in A. marginale, the msp2(p44) gene in this expression site is polymorphic in all populations of organisms we have examined, whether organisms are obtained from in vitro culture in human HL-60 cells, from culture in the tick cell line ISE6, or from infected human blood. Changes in culture conditions were found to favor the growth and predominance of certain msp2(p44) variants. Insertions, deletions, and substitutions in the region of the genomic expression site encoding the central hypervariable region matched sequence polymorphisms in msp2(p44) mRNA. These data suggest that, similarly to A. marginale, A. phagocytophilum uses combinatorial mechanisms to generate a large array of outer membrane protein variants. Such gene polymorphism has profound implications for the design of vaccines, diagnostic tests, and therapy.

Cultivation of an ovine strain of Ehrlichia phagocytophila in tick cell cultures.

Ehrlichia phagocytophila (previously known as Cytoecetes phagocytophila) which causes tick-borne fever (TBF) in sheep and pasture fever in cattle in the UK and mainland Europe is transmitted by the temperate hard tick Ixodes ricinus. The disease in sheep is characterized by fever, leucopenia and immunosuppression. Studies on the pathogenesis and other aspects of the disease have been hampered because the organism has not been cultivated in continuous or primary cell culture systems. This paper describes the first successful cultivation of a European isolate of E. phagocytophila in two continuous cell lines, IDE8 and ISE6, derived from the temperate hard tick Ixodes scapularis. Once adapted to tick cell cultures the organism was serially sub-cultured in new cells by transferring small portions of infected cell suspension every 2 to 3 weeks. The identity of the organism was confirmed by polymerase chain reaction (PCR), with primers specific to the granulocytic ehrlichiae. Sequence analysis of the PCR products amplified from infected tick cells were shown to be identical with those amplified from the blood of sheep infected with the same strain of E. phagocytophila. A susceptible sheep inoculated with a third passage of the tick cell-adapted E. phagocytophila reacted with fever and rickettsiaemia 5 days later, thus satisfying Koch's postulates.

Host cell-specific expression of a p44 epitope by the human granulocytic ehrlichiosis agent.

The human granulocytic ehrlichiosis agent (HGEa) survives extreme differences between ticks and humans, possibly by use of differential expression of specific antigens for survival in different hosts. The role of the immunodominant p44 antigens is unknown. In this study, HGEa cultured in human or tick cells was probed with human, mouse, and hamster serum and with monoclonal antibodies (MAbs). p44 antigens were strongly expressed in human HL-60 cells but were strikingly reduced in tick cells. In HGEa alternately grown in HL-60 or tick cells, a p44 epitope recognized by MAb R5E4 was expressed in human but not tick cells. This was not a temperature effect, because incubation of infected tick cells at 37 degrees C did not induce expression of the p44 epitope. The p44 antigen predominates in human but not tick cells and may be involved in regulatory changes that mediate survival of the HGEa by immune modulation after tick transmission.

Tissue diagnosis of Ehrlichia chaffeensis in patients with fatal ehrlichiosis by use of immunohistochemistry, in situ hybridization, and polymerase chain reaction.

In the United States, human ehrlichiosis is a complex of emerging tick-borne diseases caused by 3 distinct Ehrlichia species: Ehrlichia chaffeensis, Ehrlichia ewingii, and the human granulocytotropic ehrlichiosis agent. Ehrlichioses are characterized by a mild to severe illness, and approximately 4% of cases are fatal. Because these obligate intracellular bacteria are difficult to resolve with routine histologic techniques, their distribution in tissues has not been well described. To facilitate the visualization and detection of ehrlichiae, immunohistochemistry (IHC), in situ hybridization (ISH), and polymerase chain reaction (PCR) assays were developed by use of tissues from 4 fatal cases of E. chaffeensis infection. Evidence of E. chaffeensis via IHC, ISH, and PCR was documented in all 4 cases. Abundant immunostaining and in situ nucleic acid hybridization were observed in spleen and lymph node from all 4 patients. Significantly, in 2 of these patients, serologic evidence of infection was absent. Use of IHC, ISH, and PCR to visualize and detect Ehrlichia in tissues can facilitate diagnosis of ehrlichial infections.

Isolation of a spotted fever group Rickettsia, Rickettsia peacockii, in a Rocky Mountain wood tick, Dermacentor andersoni, cell line.

An embryonic cell line (DAE100) of the Rocky Mountain wood tick, Dermacentor andersoni, was observed by microscopy to be chronically infected with a rickettsialike organism. The organism was identified as a spotted fever group (SFG) rickettsia by PCR amplification and sequencing of portions of the 16S rRNA, citrate synthase, Rickettsia genus-specific 17-kDa antigen, and SFG-specific 190-kDa outer membrane protein A (rOmpA) genes. Sequence analysis of a partial rompA gene PCR fragment and indirect fluorescent antibody data for rOmpA and rOmpB indicated that this rickettsia was a strain (DaE100R) of Rickettsia peacockii, an SFG species presumed to be avirulent for both ticks and mammals. R. peacockii was successfully maintained in a continuous culture of DAE100 cells without apparent adverse effects on the host cells. Establishing cell lines from embryonic tissues of ticks offers an alternative technique for isolation of rickettsiae that are transovarially transmitted.

Growth of Cowdria ruminantium, the causative agent of heartwater, in a tick cell line.

The tick-borne rickettsia Cowdria ruminantium has been propagated continuously for over 500 days in the Ixodes scapularis tick cell line IDE8 by using the Gardel isolate from bovine endothelial cells as an inoculum. Infection of the tick cells was confirmed by PCR, karyotyping, electron microscopy, and reinfection of bovine cells.

Differential expression of outer surface proteins A and C by individual Borrelia burgdorferi in different genospecies.

Previous studies with different Borrelia burgdorferi sensu stricto (s.s.) strains revealed that temperature as well as cocultivation with tick cells modulates the expression of outer surface proteins (Osp) A and C. We investigated the effects of temperature and of interaction with tick cells in culture on the expression of OspA and OspC of the B. afzelii clones cPKo97 and cPKo345 in comparison to the B. burgdorferi s.s. strain N40. To follow the dynamics of Osp expression of single borreliae we used indirect immunofluorescence microscopy with double staining of OspA and OspC. Clone PKo345 always showed expression of only OspA, regardless the conditions it was subjected to. Sequencing of the ospC gene disclosed a insertion leading to a stop codon after base 222 and inability to produce OspC. In cPKo97 and N40 OspC is down-regulated at lower temperatures and up-regulated at higher temperatures, which was especially pronounced on cocultivation with tick cells. Borreliae adherent to tick cells showed greater OspA expression compared to the nonadherent ones, an indication that OspA might play a role as adhesin for tick cells. Interestingly, cPKo97 and N40 displayed different patterns of Osp expression: cPKo97 simultaneously presents OspA and OspC on single borreliae, while N40 has either OspA or OspC on single cells. Adaptation of OspC expression in cPKo97 seems to occur by up- or down-regulation of this protein on single borreliae, as shown by alternating intensities of OspC expression at different temperatures. In contrast, N40 seem to consist of two subsets of borreliae one expressing only OspA and the other only OspC, and change in temperature results in growth benefit for one of these subtypes. Our findings indicate that, regarding OspA and OspC expression, response to temperature and cocultivation with tick cells of B. afzelii is comparable to B. burgdorferi s.s., but the mode of regulation seems phenotypically different. Further European isolates should be investigated for OspA and OspC regulation, especially in the face of vaccine development for the European situation.

Invasion and intracellular development of the human granulocytic ehrlichiosis agent in tick cell culture.

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

Borrelia burgdorferi in tick cell culture modulates expression of outer surface proteins A and C in response to temperature.

The Lyme disease spirochete Borrelia burgdorferi sensu stricto downregulates outer surface protein A (OspA) and upregulates outer surface protein C (OspC) during tick feeding. The switching of these proteins correlates with increased spirochetal infectivity for the mammal. We examined the effect of temperature on differential expression of OspA and OspC by B. burgdorferi cocultivated with a cell line isolated from the vector tick Ixodes scapularis. The effect of incubation at 31, 34, or 37 degrees C on expression of OspA and OspC by B. burgdorferi JMNT and N40 was analyzed by indirect fluorescent-antibody microscopy, polyacrylamide gel electrophoresis, and immunoblotting. The amount of OspA relative to the amount of flagellin was highest in spirochetes cocultivated with tick cells at 31 degrees C and declined with increasing temperature in both strains. OspC production was enhanced in spirochetes cocultivated with tick cells at 37 degrees C. Spirochetes grown axenically in BSK-H medium also produced more OspC at 37 degrees C, but OspA content was not appreciably affected by temperature. Our findings indicate that temperature, along with cultivation in a tick cell culture system, plays a role in the differential expression of OspA and enhances differential expression of OspC by spirochetes.

Epidemiological aspects of human granulocytic Ehrlichiosis in southern Germany.

Human granulocytic Ehrlichiosis (HGE) is a newly emerging acute febrile illness which is likely transmitted by ticks of the Ixodes ricinus/I. persulcatus complex. First seroepidemiological surveys on the prevalence of HGE antibodies, detection of DNA of granulocytotropic Ehrlichiae in I. ricinus and one case of HGE from Slovenia confirmed by serology and PCR (polymerase chain reaction) suggest that HGE might exist all over Europe. The purpose of the present study was a) to determine the prevalence of antibodies against the HGE agent in sera collected from persons at high risk for exposure to I. ricinus with that of a control population and b) to determine the prevalence of granulocytic Ehrlichiae in I. ricinus ticks from Southern Germany. We studied sera from 150 forestry workers and 105 patients with an established diagnosis of Lyme disease as tick-exposed populations. Sera from 103 healthy blood donors without a history of known tick bites served as controls. A significantly higher prevalence of HGE antibodies (P < or = 0.01) was present among patients with Lyme borreliosis (12 of 105 were positive; 11.4%) and forestry workers (21 of 150 were positive; 14%) compared to blood donors (2 of 103 were positive; 1.9%). Furthermore, 510 adult and nymphal I. ricinus were investigated by PCR for the presence of granulocytic Ehrlichiae with primers specific for the E. phagocytophila group. In eight (1.6%) of the investigated ticks the expected amplification product was detectable, indicating a low prevalence of infected ticks especially when compared with B. burgdorferi. The presented data strongly suggests that the HGE agent or a closely related organism exists in Southern Germany and therefore HGE should be considered in the differential diagnosis of febrile illnesses. However, final evidence can be provided only after isolation of the organism from patients.

Coexistence of ehrlichiae of the phagocytophila group with Borrelia burgdorferi in Ixodes ricinus from Southern Germany.

Human granulocytic ehrlichiosis (HGE) is an emerging infectious disease recognized in the Western hemisphere. HGE is well known to occur in North America, but records from outside the United States are sparse. The great majority of data from Europe are restricted to seroprevalence studies and molecular biological detection of granulocytic ehrlichiae (GE) in ticks and mammals, but include defined cases from Slovenia. They argue for the existence of this disease in many parts of Europe. In the present study, 510 Ixodes ricinus ticks collected in five different regions of Southern Germany were investigated for the presence of GE and Borrelia burgdorferi sensu lato using polymerase chain reaction. In all, 8 (1.6%) of the 492 ticks that could be evaluated (193 females, 208 males, and 91 nymphs) contained GE and 178 (36.2%) B. burgdorferi s.l.. Four of these ticks were infected with both pathogens. Interestingly, all ehrlichia-infected ticks were adults and all were collected in the English Garden, a recreational park area located in the city of Munich. Sequencing of the 16S rDNA (bp 1 1101) of four of the GE showed 100% sequence identity to each other and greater than 99.9% identity with the published sequence of the HGE agent. The four GE differed in respect to other hitherto described GE by a nucleotide exchange at position 336. These results show that GE that are closely related to the HGE agent are present in Southern Germany, and that coinfection with B. burgdorferi is common in GE-infected ticks. However, in contrast to B. burgdorferi which is endemic everywhere in Southern Germany, the distribution of GE seems to be focal.

Phylogenetic placement of rickettsiae from the ticks Amblyomma americanum and Ixodes scapularis.

A rickettsial isolate (isolate MOAa) belonging to the spotted fever group (SFG) was obtained from the lone star tick Amblyomma americanum. We used PCR to characterize the genes for the rickettsial outer membrane proteins rOmpA and rOmpB. We sequenced the PCR products (domains I of both the rompA gene and the rompB gene) of MOAa and WB-8-2, another rickettsial isolate from A. americanum. To place MOAa and WB-8-2 and two other nonpathogenic isolates (Rickettsia rickettsii Hip2 and Rickettsia montana M5/6) with respect to their putative sister species, we included them in a phylogenetic analysis of 9 Rickettsia species and 10 Rickettsia strains. Our phylogenetic results implied three evolutionary lineages of SFG rickettsiae and that WB-8-2 and MOAa were most closely related to R. montana. New World isolates were not the most closely related to each other (they did not form a clade). Rather, our results supported four independent origins (introductions) of rickettsiae into North America from different Old World regions. The results of our phylogenetic analysis did not support the hypothesis of a stable coevolution of rickettsiae and their tick hosts. Finally, we examined the rompA gene of a nonpathogenic rickettsial symbiont isolated from the tick Ixodes scapularis. In a phylogenetic analysis, the symbiont was placed as the sister to R. montana and its isolates. The relationship of this symbiont to R. montana raised questions as to the potential origin of pathogenic SFG rickettsiae from nonpathogenic tick symbionts, or vice versa.

Human granulocytic ehrlichiosis in southern Germany: increased seroprevalence in high-risk groups.

To date, human granulocytic ehrlichiosis (HGE), the causative agent of which is likely transmitted by ticks in the Ixodes ricinus-Ixodes persulcatus complex, has not been diagnosed with certainty in patients outside the United States. The presence of a closely related vector tick, I. ricinus, as well as the occurrence of similar Ehrlichia spp. of veterinary importance, suggests that this disease is likely to be present in Europe. The aim of the present study was to compare the prevalence of antibodies against the HGE agent in sera collected from patients in groups at high risk for exposure to I. ricinus with that of a control population. Risk groups consisted of 150 forestry workers and 105 patients with an established diagnosis of Lyme disease. The control group was 103 healthy blood donors without a history of tick bites. We used a patient isolate of the HGE agent from Minnesota (J. L. Goodman, C. Nelson, B. Vitale, J. E. Madigan, J. S. Dumler, T. J. Kurtti, and U. G. Munderloh, N. Engl. J. Med. 334:209-215, 1996) propagated in HL60 cells as the source of antigen for a specific immunofluorescence assay (IFA). Elevated IFA titers (> or = 1:80) were present in 21 of 150 (14%) serum samples from forestry workers and in 12 of 105 (11.4%) serum samples from Lyme disease patients, but in only 2 of 103 (1.9%) serum samples from blood donors (P < or = 0.01 for either of the at-risk groups versus blood donors). The results of this study suggest that the HGE agent or a closely related organism exists in southern Germany and that seroconversion to it is common among groups exposed to Ixodes ticks. Final proof that HGE occurs in Germany will require the isolation of the causative agent from patients. HGE should be considered in the differential diagnosis of febrile illnesses in individuals exposed to Ixodes ticks in Europe as well as in North America.

Endosymbionts of ticks and their relationship to Wolbachia spp. and tick-borne pathogens of humans and animals.

The presence, internal distribution, and phylogenetic position of endosymbiotic bacteria from four species of specific-pathogen-free ticks were studied. These included the hard ticks Ixodes scapularis (the black-legged tick), Rhipicephalus sanguineus (the brown dog tick), and Haemaphysalis longicornis and the African soft tick Ornithodoros moubata. PCR assays for bacteria, using two sets of general primers for eubacterial 16S and 23S rRNA genes (rDNAs) and seven sets of specific primers for wolbachial, rickettsial, or Francisella genes, indicated that I. scapularis possessed symbiotic rickettsiae in the ovaries and that the other species harbored eubacteria in both the ovaries and Malpighian tubules. Phylogenetic analysis based on the sequence of 16S rDNA indicated that the symbiont of I. scapularis belonged to the alpha subgroup of proteobacteria and was closely related to the members of the genus Rickettsia. The other species had similar microorganisms in the ovaries and Malpighian tubules, which belonged to the gamma subgroup of proteobacteria, and formed a monophyletic group with the Q-fever pathogen, Coxiella burnetii. O. moubata harbored another symbiont, which formed a monophyletic group with Francisella tularensis and Wolbachia persica, the latter a symbiont previously isolated from Malpighian tubules of the soft tick Argas (Persicargas) arboreus. Thus, the symbionts of these four tick species were not related to the Wolbachia species found in insects. The two symbionts that live in the Malpighian tubules, one closely related to C. burnetii and the other closely related to F. tularensis, appear to be of ancient origin and be widely distributed in ticks.

Rickettsia rickettsii growth and temperature-inducible protein expression in embryonic tick cell lines.

Rickettsia rickettsii has limited adverse effects on its arthropod vector, but causes severe disease in man. To model differences in host-parasite interaction, R. rickettsii growth and protein expression were examined at temperatures reflective of host environment in the tick cell lines DALBE3 and IDE2, the human endothelial cell line ECV304, and the African green monkey kidney cell line Vero76. At low multiplicities of infection, rickettsial titres increased 10(2)-10(3)-fold in all cell lines after incubation for 3 days at 34 degrees C. At higher multiplicities and with extended incubation, R. rickettsii showed enhanced survival in tick versus mammalian cells. No difference in rickettsial ultrastructure or protein profiles was detected between different host cell types. Rickettsial proteins of 42, 43, 48, 75 and 100 kDa are induced in tick cells shifted from 28 degrees to 34 degrees C, but not in cells maintained at 28 degrees C. This temperature response may be associated with expression of rickettsial determinants that are pathogenic to mammalian hosts.

Resistance to tick-borne spirochete challenge induced by Borrelia burgdorferi strains that differ in expression of outer surface proteins.

Hamsters were immunized with thimerosal-killed Borrelia burgdorferi 297 or a mutant of 297 (M297) that lacks the 49-kb linear plasmid and expression of outer surface proteins A and B (OspA and OspB). Ixodes scapularis nymphs infected with either the B. burgdorferi sensu stricto strain 297 or JMNT, similar in OspA and OspB but differing in OspC expression, were used to evaluate protection. In a homologous challenge, 24 hamsters were vaccinated, 8 each with 297 or M297 and 8 sham (adjuvant)-vaccinated controls. Hamsters vaccinated with either bacterin were completely protected against a natural tick bite or subcutaneous (s.c.) inoculation of 297. Borreliae were effectively eliminated from 80 to 90% of the 297-infected ticks that fed on four hamsters immunized with the 297 bacterin. Cultures of spirochetes isolated from the ticks that remained infected were infectious and induced joint inflammation in naive hamsters. There was no reduction of strain 297 spirochetes in ticks that fed on four hamsters immunized with M297, but the hamsters were protected. Results with the M297 bacterin indicate that proteins other than OspA or OspB can protect hamsters against a tick challenge without eliminating B. burgdorferi in the tick. In a heterologous challenge, 36 hamsters were vaccinated, 12 with each bacterin and 12 controls. None of the hamsters immunized with either bacterin were protected from a challenge involving JMNT-infected ticks, while two of four were protected against an s.c. challenge. Hamsters challenged s.c. with strain 297 spirochetes were protected. There was partial elimination of JMNT spirochetes in ticks that fed on the group of four hamsters immunized with the 297 bacterin, and infection rates were reduced by 50 to 60%. JMNT spirochetes reisolated from the ticks that fed on 297-vaccinated hamsters also remained infectious for hamsters. In the JMNT-infected ticks that fed on four M297-immunized hamsters, there was no decline in the proportion of infected ticks. Destruction of spirochetes in ticks that fed on the hamsters vaccinated with the 297 bacterin suggests that antibodies to OspA and OspB may have been responsible, since the mutant did not induce this activity.

Establishment of the tick (Acari:Ixodidae)-borne cattle pathogen Anaplasma marginale (Rickettsiales:Anaplasmataceae) in tick cell culture.

Anaplasma marginale is a tick-borne rickettsia that causes bovine anaplasmosis worldwide. Despite its importance, A. marginale has thus far not been established in a continuous culture system. We have propagated A. marginale continuously for the 1st time in a tick cell line derived from the black-legged tick, Ixodes scapularis Say, using infected bovine blood as the inoculum. Erythrocytic stages invaded the tick cells and multiplied in membrane-lined vacuoles to form colonies typical of those observed in naturally infected ticks as demonstrated by light and electron microscopy. The rickettsiae have been passaged serially for 3 yr and have been cryopreserved in liquid nitrogen. Antigens present in A. marginale from tick cell culture were recognized by bovine immune serum against the blood stages of A. marginale. A. marginale grown in this tick cell line was infective for calves, and male ticks fed on the calves transmitted A. marginale to a susceptible calf. The ability to culture A. marginale removes a major impediment to the study of Anaplasma biology in vitro, and will enhance development of vaccines and diagnostic tests.

Analysis of linear plasmid dimers in Borrelia burgdorferi sensu lato isolates: implications concerning the potential mechanism of linear plasmid replication.

The Borrelia genome is composed of a linear chromosome and a number of variable circular and linear plasmids. Atypically large linear plasmids of 92 to 105 kb have been identified in several Borrelia burgdorferi sensu lato isolates and characterized. These plasmids carry the p27 and ospAB genes, which in other isolates reside on a 50-kb plasmid. Here we demonstrate that these plasmids are dimers of the 50-kb ospAB plasmid (pAB50). The 94-kb plasmid from isolate VS116, pVS94, was an exception and did not hybridize with any plasmid gene probes. When this plasmid was used as a probe, homologous sequences in other isolates were not detected, suggesting that it is unique to isolate VS116. These analyses provide insight into the mechanism of linear plasmid replication and the mechanisms by which plasmid variability can arise.

Isolation of the equine granulocytic ehrlichiosis agent, Ehrlichia equi, in tick cell culture.

The equine granulocytic ehrlichiosis agent, Ehrlichia equi, is closely related or identical to the human granulocytic ehrlichiosis (HGE) agent. Both are suspected of being transmitted by ticks. We have successfully isolated E. equi in a cell line, IDE8, derived from a putative vector, the tick Ixodes scapularis. Peripheral blood leukocytes from an experimentally infected horse were inoculated onto IDE8 monolayers. Cultures were incubated in a candle jar at 34 degrees C in tick cell culture medium with NaHCO3 and an organic buffer [3-(N-morpholino)-propanesulfonic acid] (MOPS). Within 2 weeks, infected cells were detected in Giemsa-stained culture samples, and the organisms subsequently spread to uninfected cells in the cultures. E. equi was passaged serially by transferring a portion of an infected culture to new cell layers every 2 to 3 weeks. The identity of the organisms was confirmed by PCR using oligonucleotide primers specific for E. equi and the HGE agent and by immunocytology. Homologous equine antibodies and human anti-HGE convalescent serum recognized E. equi grown in tick cell culture. Electron microscopy revealed electron-lucent and -dense ehrlichia-like forms developing within host cell endosomes. E. equi passaged twice in tick cell culture retained infectivity and pathogenicity for the equine host, as demonstrated by intravenous inoculation of a suspension of infected tick cells and subsequent reisolation from peripheral blood, in fulfillment of Koch's postulates. The horse developed severe clinical signs, i.e., fever, inappetence, thrombocytopenia, icterus, and limb edema, typical of granulocytic equine ehrlichiosis, within 1 week.