Cultivation of an Ovine Strain of Ehrlichia phagocytophila in Tick Cell Cultures

J. Comp. Path. 2002, Vol. 127, 142±149 doi:10.1053/jcpa.2002.0574, available online at http://www.idealibrary.com on

Cultivation of an Ovine Strain of Ehrlichia phagocytophila in Tick Cell Cultures Z. Woldehiwet*, B. K. Horrocks*, H. Scaifey, G. Ross*, U. G. Munderlohz, K. Bown{, S. W. Edwardsy and C. A. Hartx *Department of Veterinary Pathology, ySchool of Biological Sciences, xDepartment of Medical Microbiology and {Department of Clinical Veterinary Sciences, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, UK and zDepartment of Entomology, University of Minnesota, St Paul, Minnesota, USA Summary 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. # 2002 Elsevier Science Ltd. All rights reserved.

Introduction Ehrlichia phagocytophila (previously known as Cytoecetes phagocytophila), the causative agent of tick-borne fever ( TBF ) in sheep and pasture fever in cattle, is an obligately intracellular bacterial pathogen whose known mammalian target cells are mainly peripheral blood granulocytes, although the organism can also infect monocytes (Woldehiwet and Scott, 1993). It has recently been proposed that E. phagocytophila, E. equi and the agent of human granulocytic ehrlichiosis (aoHGE) be united with the genus Anaplasma, together with Ehrlichia bovis and Ehrlichia platys (Dumler et al., 2001). The disease, which is transmitted by the temperate tick Ixodes ricinus, is common in Scotland (Gordon et al., 1932) and northern and southwest England and Wales. The disease is also widespread in Scandinavia (Tuomi, 1967) and other parts of mainland Europe (Woldehiwet and Scott, 1993). In the 0021±9975/02/$ ± see front matter

United States the principal vectors for E. equi and for aoHGE are Ixodes scapularis and Ixodes pacificus (Richter et al., 1996; Telford et al., 1996). The target cell specificity for granulocytes and monocytes has hampered efforts to cultivate E. phagocytophila in vitro. Recent studies have shown that the monocytic ehrlichiae can be cultivated in cell lines from their natural mammalian hosts and in other established mammalian cell lines. For example, Ehrlichia chaffeensis has been cultivated in mouse embryo, Vero, BGM and L929 cell lines (Chen et al., 1995). Ehrlichia equi, the causative agent of equine ehrlichiosis, and aoHGE have been cultivated recently in the human promyelocytic leukaemia cell line HL-60 (Goodman et al., 1996). Both agents have also been cultivated in tick cell culture systems (Munderloh et al., 1996a, 1999). However, to our knowledge the agents that cause TBF (ovine and bovine strains of E. phagocytophila) and # 2002 Elsevier Science Ltd. All rights reserved.

Cultivation of E. phagocytophila

bovine petechial fever (Cytoecetes ondiri) have not been successfully cultivated in vitro, other than for short-term purposes (Woldehiwet and Scott, 1982a; Jongejan et al., 1989). Early attempts to grow E. phagocytophila in mammalian cells (Tuomi, 1967; Thrusfield et al., 1978) and in organ cultures from its tick vector (Woldehiwet, 1981) were not successful. The present study reports the successful cultivation of an ovine (Old Sourhope) strain of E. phagocytophila in vitro in two embryonic tick cell lines, IDE8 and ISE6, derived from Ixodes scapularis. The same cell lines have been shown to support the growth of the genetically related organisms E. equi (IDE8, Munderloh et al., 1996a). Anaplasma marginale (Munderloh et al., 1996b) and aoHGE (ISE6, Munderloh et al., 1999). Materials and Methods Tick Cell Cultures The tick cell lines IDE8 (ATCC CRL, 11973) and ISE6 (Munderloh, 1999), both isolated from embryos of Ixodes scapularis, were maintained in L-15B medium, pH 7
2, supplemented with heat-inactivated fetal calf serum (FCS) (Sigma Aldrich, Poole, Dorset, England), tryptose phosphate broth (ICN Pharmaceuticals, Basingstoke, England) 10% and bovine lipoprotein concentrate (ICN Pharmaceuticals) 0
1% as described by Munderloh and Kurtti (1989) and Munderloh et al. (1994). Cell monolayers were propagated in 25-cm2 plastic flasks or flat-sided tissue culture tubes (Nalge Nunc International, Roskilde, Denmark) and the IDE8 cells and ISE6 cells were maintained at 32 C and 34 C, respectively (Munderloh et al., 1999). Confluent monolayers were kept for up to 6 weeks by replacing one-third of the conditioned medium with new medium weekly. Inoculation of Tick Cells with E. phagocytophila The inoculum for tick cells was heparinized ovine blood obtained on the second day of bacteraemia of a sheep infected with the Old Sourhope (OS) strain of E. phagocytophila, as described previously (Woldehiwet, 1987). The whole blood was diluted 1 in 4 with sucrose phosphate glutamate buffer (Woldehiwet et al., 1991) and 0
2 ml and 0
05 ml of this suspension were then added to monolayers of tick cells in 25-cm2 plastic flasks and tissue culture tubes, respectively. Tick cells were maintained in L-15B medium with penicillin 100 iu/ml and streptomycin 100 m/ml until they became confluent. Immediately before inoculation, the medium was decanted and replaced with new pre-warmed medium, without antibiotics but

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supplemented with NaHCO3 0
25% and 25 mM HEPES at pH 7
5. After inoculation, all cells were incubated at 34 C. Vented flasks were kept in candle jars or in an incubator with CO2 4%. Sealed flasks and culture tubes were kept in separate incubators with atmospheric air. The cells were maintained by replacing one-third of the conditioned medium with new medium weekly. Serial passages were carried out every 3±4 weeks by adding 0
1 to 0
2 ml of medium from infected flasks or tubes to uninfected monolayers in flasks or tubes. Microscopy Small volumes (0
2±0
5 ml) were removed every 3±4 days and deposited on microscope slides with a Cytospin centrifuge (Shandon, Runcorn, Cheshire, UK). The cells were air-dried, fixed in methanol for 5 min and then stained in May and Grunwald's solution (in Sorensen buffer) for 5 min and Giemsa stain in Sorenson buffer (BDH, Poole, Dorset, England) for 10 min. The cells were then differentiated in Sorenson's buffer (pH 6
8) for 5 min, air-dried and examined under a light microscope. Ultrastructure Control and infected tick cells were prepared for electron microscopy as described previously (Woldehiwet and Scott, 1982b), with some modifications. Briefly, cell pellets were fixed with a mixture of formaldehyde 1
25%, glutaraldehyde 2
5% and trinitrophenol 0
03% in 0
1 M cacodylate buffer (pH 7
4) for 24 h. Cells were then post-fixed in osmium tetraoxide 1% (Taab Laboratories, Reading, UK) in veronal-acetate buffer solution (Sigma) for 1 h and dehydrated by immersing them in increasing concentrations of acetone ranging from 50 to 100%. Cell pellets were embedded in Spurr's resin and ultrathin sections were stained with uranyl acetate (Taab Laboratories) and Reynold's lead citrate. Finally the sections were examined with an electron microscope (Model H-600; Hitachi, Tokyo, Japan). Infectivity and Pathogenicity for Sheep After the third passage in IDE8 cells, the tick-celladapted isolate was tested for infectivity and pathogenicity in a susceptible sheep. A 4-year-old Suffolk cross sheep, bred and raised under tick-free conditions, was inoculated intravenously with 1 ml of infected tick cell suspension derived from a flask showing evidence of cytopathogenic effects (cpe) and high rates of

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infection, 3 weeks after the third passage. Rectal temperatures were taken daily and the animal was monitored for clinical signs and bacteraemia, as described earlier (Woldehiwet, 1987). Blood samples were collected in EDTA-coated tubes for total and differential white cell counts and to prepare smears. The smears were stained with Giemsa and examined for inclusions in granulocytes and monocytes, as described by Woldehiwet (1987). Blood samples obtained before and after inoculation were also tested for the presence of bacterial DNA by PCR, as described below. Serum samples were collected before inoculation and every week thereafter for 4 weeks, and stored at ÿ20 C. The sera were inactivated (30 min at 56 C), adsorbed with uninfected tick cells by an overnight incubation at 4 C, and then tested for specific antibodies with infected ovine granulocytes or infected tick cells as antigens, as described by Gocke and Woldehiwet (1999). Briefly, infected tick cells and ovine granulocytes were fixed in a 1 : 1 mixture of methanol and acetone and stored at ÿ20 C until required. Serum samples were diluted 1 in 80 and used as primary antibody, and fluorescein isothiocyanate (FITC)-conjugated rabbit anti-sheep IgG (Sigma) was used as secondary antibody. Normal ovine granulocytes and uninfected tick cells were used as negative control antigens. Serum obtained from the same sheep before inoculation was used as a negative serum control. The slides were counterstained with Evans blue, mounted with glycerol 10% in phosphate-buffered saline and examined under an immunofluorescence microscope (Leitz Divert, Midland, Ontario, Canada).

Polymerase Chain Reaction (PCR) DNA was extracted from infected IDE8 and ISE6 tick cells, at the 6th passage, as described previously (Ogden et al., 1998) and amplified by PCR, with the species-specific primers GER3 (50 TAG ATC CTT CTT AAC GGA AGG GCG 30 ) and GER 4 (50 AAG TGC CCG GCT TAA CCC GCT GGC 30 ). These primers correspond to bases 950±973 and 1077±1101 of the 16s rRNA gene, respectively, and amplify a 151-bp fragment from the granulocytic ehrlichiae (E. phagocytophila, E. equi and aoHGE) but not from monocytic ehrlichiae (Goodman et al., 1996). For comparison, DNA was extracted from whole ovine blood infected with the same strain of E. phagocytophila. Uninfected IDE8 and ISE6 cells and uninfected sheep blood were used as negative controls. Amplification was carried out in a thermal cycler (Hybaid, Ashford, Middlesex, England) with 5 min of pre-heating at

95 C, followed by 40 cycles consisting of 1 min of denaturing at 94 C, 1 min of annealing at 50 C, and 1 min of extension at 72 C for all cycles but the 40th, in which the extension lasted for 7 min. Cloning and Sequencing of PCR Products The 16S rDNA to be sequenced was amplified from infected tick-cells by means of a nested PCR (Barlough et al., 1996) with the outer primers EE-1 (50 TCC TGG CTC AGA ACG AAC GCT GGC GGC 30 ) and EE-2 (50 AGT CAC TGA CCC AAC CTT AAA TGG CTG 30 ) and the inner primers EE-3 (50 GTC GAA CGG ATT ATT CTT TAT AGC TTGC 30 ) and EE-4 (50 CCC TTC CGT TAA GAA GGA TCT AAT CTCC 30 ). Amplification was carried out in a thermal cycler (Hybaid) with 5 min of pre-heating at 94 C and 35 cycles consisting of denaturing at 94 C, followed by 2 min of annealing at 50 C, and 30 s of extension at 72 C for all cycles and for each round. These primers span bases 1±27 (EE-1), 1433±1407 (EE-2), 45±72 (EE-3) and 970±943 (EE-4) of the reported 16S rDNA sequence for aoHGE (GenBank accession No. U02521). The strands of the amplification product were then cloned by means of the TOPOTAC cloning kit with vector pCR4 (Invitrogen, Paisley, Scotland) as per manufacturer's instructions. The products were then sequenced with an ABI 377 automated sequencer (Applied Biosystems, Foster City, USA). Results Isolation of E. phagocytophila Evidence of infection was first detected in IDE8 and ISE6 cells at 7 days post-inoculation (pi). However, the percentage of infected cells was low (less than 1 in 500 cells were positive). The prevalence of infection continued to rise slowly, reaching 15% by 6 weeks pi. The first successful subculture was carried out 6 weeks after primary culture, but subsequent subcultures could be established as early as 2 weeks after primary culture in both cell types. At the time of writing, E. phagocytophila had been successfully passaged 12 times. The organisms were capable of infecting both cell lines maintained in vented flasks in 4% CO2, in a candle jar, or in sealed flasks or tubes. However, because the cells did not tolerate the CO2 atmosphere well, all subsequent primary cultures and passages were carried out in sealed flasks or cell culture tubes. Cytopathogenic effects characterised by the enlargement, detachment and lysis of infected cells were first observed 4±6 weeks after inoculation with infected blood, with the cpe becoming apparent 2±3 weeks

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after subculture. Not all the enlarged and vacuolated cells harboured inclusions. No distinct difference in the nature of the cpe was observed in the two cell lines used. Infected IDE8 cells were successfully stored in liquid nitrogen, with DMSO 10% in tick cell growth medium (with 20% FCS) as a cryopreservative. IDE8 and ISE6 cells inoculated with the stabilates developed typical inclusions within 2 weeks and were successfully subcultured. Infection was also successfully initiated in new IDE8 cells with cell-free tick cell-derived E. phagocytophila. Morphological Features of E. phagocytophila in Tick Cell Cultures In tick cells the ehrlichiae were quite distinct in appearance from the morula-like inclusions commonly found in ovine peripheral neutrophils. With Giemsa they were stained an intense purple-blue and were ``inklike'' in heavily infected cells, in which they often occupied nearly the whole cell with only a small remaining amount of host cell cytoplasm, adjacent to the nucleus (Figs 1a, b). They appeared as small or large clusters of organisms with discrete particles or as compact colonies, without discernible particles, either in the periphery of the cytoplasm or over the whole cell. Some cells contained several inclusions and on occasion large clusters of organisms were present in cells that had completely lost their integrity (not shown). Not surprisingly, E. phagocytophila in tick cells appeared to have the same staining characteristics as E. equi (Munderloh et al., 1996a). Often the structures described above were not clearly associated with well-defined cells.

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Ultrastructure in Tick Cells Infected tick cells contained pleomorphic bacterial structures of varying sizes, shapes and electron density (Fig. 2). In heavily infected cells, a small section of host cytoplasm on the periphery of the nucleus was pushed aside, with the ehrlichiae occupying nearly the whole cell (Fig. 2). Some inclusions contained only electronlucent structures while others contained electrondense structures in addition (Figs 2a, b). On a few occasions the electron-dense structures appeared to be within larger electron-lucent structures (Fig. 2c), but the former may have been merely pouched within a fold of the latter. On some occasions, the ehrlichiae were also frequently found outside intact cells, as the tick cells lost their integrity due to infection (not shown); most of these structures were electron-lucent, only a few being electron-dense structures. Infectivity and Pathogenesis The sheep infected with the tick-cell adapted isolate of E. phagocytophila reacted with a fever, bacteraemia and leucopenia typical of TBF, starting at 5 days postinoculation (dpi). The febrile reaction lasted for 5 days and the organism was detected in peripheral blood for up to 10 dpi. The presence of the organism in peripheral blood was also confirmed by PCR in samples taken at 6 dpi. Antibodies specific to E. phagocytophila were detected in serum samples obtained from the sheep at 14±28 dpi by indirect immunofluorescence with infected ovine granulocytes or infected tick cells as antigens. Two sheep inoculated with control IDE8 cells showed no reaction.

Fig. 1a, b. Ehrlichia phagocytophila colonies in IDE8 cells. The ehrlichiae appear as large colonies or small discrete particles. (a) Cells harbouring large dispersing (LD) colonies and compact dense (CD) colonies. N, host cell nucleus. Giemsa. 900. (b) Hypertrophied tick cell over-laden with large dense colonies. N, host cell nucleus. Giemsa. 900.

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Fig. 2a±c. Ehrlichia phagocytophila in IDE8 cells. (a) Colonies with electron-lucent organisms. Electronmicroscopy (EM). 7200. (b) Inclusion containing a mixture of electron-dense and electron-lucent particles. EM. 540. (c) Inclusion with electrondense particles apparently developing or pouched within folds of electron-lucent vegetative forms. EM. 7200.

PCR and Sequence Analysis Primers GER3 and GER4 (Goodman et al., 1996), which are specific to the 16S rRNA genes of the granulocytic ehrlichiae, E. phagocytophila, E. equi and aoHGE, amplified a product which was 151 bp in size from IDE8 tick cell cultures infected with E. phagocytophila, but not from the uninfected controls. The primers also amplified an identical PCR product from the blood of the sheep with rickettsiaemia but not from control ovine blood. A 928-bp product was generated from the tick celladapted isolate and from infected ovine granulocytes with E. phagocytophila-specific nested PCR (Barlough et al., 1996). Sequence analysis of the cloned 928-bp PCR products generated by nested PCR revealed a 100% identity between the tick-cell-adapted strain

and the original OS strain of E. phagocytophila. The two products had 100% identity with the published sequences of the OS and Feral Goat (FG) strains of E. phagocytophila (GenBank accession number M73220 and M73224). Discussion Tick-borne fever was the first disease of animals to be associated with a granulocytic ehrlichia. In an experiment designed to investigate the transmission of louping-ill virus by ticks, research workers in Scotland (Gordon et al., 1932) discovered that the hard tick, I. ricinus, was apparently harbouring an agent different from the virus that caused louping ill. After the demonstration of the causative agent in peripheral

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blood granulocytes and monocytes (Gordon et al., 1940), concerted efforts were made to grow the organisms in vitro, without success. Early workers attempted to grow the organism in simple bacteriological media (Gordon et al., 1932; Tuomi, 1967) and in embryonated eggs (Hudson, 1950; Tuomi, 1967). After the recognition that the organism was an intracellular pathogen, a few workers have reported unsuccessful attempts to cultivate it in primary and continuous mammalian cell lines (Tuomi, 1967; Thrusfield et al., 1978). The main obstacle to successful cultivation was believed to be the organism's high degree of target cell specificity (Thrusfield et al., 1978). It was recognized that the most likely targets would be cells of the phagocytic or endothelial system of the mammalian host, or cells derived from the arthropod vector (Woldehiwet, 1981). However, attempts to grow the organism in organ cultures of the hard tick I. ricinus and ovine monocytic cell lines were not successful (Woldehiwet, 1981), but the organism was shown to continue to multiply in ovine and bovine granulocytes obtained from infected animals (Woldehiwet and Scott, 1982a; Jongejan et al., 1989). These short-term cultures have been successfully used to generate antigens, but granulocytes are not suitable for long-term culture because they have a short life span in the peripheral blood. This is in sharp contrast to the monocytic ehrlichiae, which are readily cultivated in cell lines derived from their natural vertebrate hosts. For example, Ehrlichia canis and E. chaffeensis can be cultivated in primary or continuous monocytic/macrophage cell lines (Nyindo et al., 1971; Dawson et al., 1991, 1993). Similarly Cowdria ruminantium, the causative agent of heartwater in ruminants, was cultivated in primary bovine endothelial cells, its natural target cells (Byron and Yanker, 1990). Subsequently, E. equi and aoHGE were cultivated in the promyelocytic cell line HL-60 (Goodman et al., 1996) and in continuous tick cell lines (Munderloh et al., 1996a, 1999). The present study marks an important development in the long search for appropriate targets for the cultivation of E. phagocytophila in vitro. Its successful cultivation was confirmed by the demonstration of typical organisms by light and electron microscopy, the demonstration of specific antigens, and the amplification of DNA specific to the E. phagocytophila genogroup. We have shown that the tick cell-adapted organism retains its infectivity and pathogenicity for sheep, thus satisfying Koch's postulates. The association of E. phagocytophila with ticks has been recognized for over 70 years, since TBF was first described in Scotland in the 1930s. However, little is known about the biology of the organism in the tick host. The present study showed that the ultrastructure

of the organisms in tick cells was different from that observed in ovine granulocytes. If the structures seen in tick cultures were the same as those found in the tick vector, it would suggest that the organisms undergo stages of development in the tick different from those in their mammalian host. The poor recognition of antigens expressed in tick cells by E. phagocytophilaspecific immune serum raised in sheep inoculated with infected blood stabilate (data not shown) may also indicate that the predominant antigens recognized by the mammalian host are not necessarily expressed in the arthropod host. Recent studies in aoHGE suggest that the predominant surface antigen, P44, was strongly expressed in HL-60 cells but much less so in tick cells ( Jauron et al., 2001). On the basis of their similarities in the sequences of their 16S ribosomal DNA and their predilection for granulocytes, E. equi, E. phagocytophila and aoHGE are widely regarded as strains of the same species (Goodman et al., 1996). However, important biological differences remain. These include mammalian host specificity. For example, attempts to transmit infection to a horse by inoculating blood obtained from infected cattle were unsuccessful (Tuomi, 1967) and there is little documented evidence of human infection in the United Kingdom, where TBF has been known for 70 years. Therefore, the biological differences between geographically different isolates of E. phagocytophila and other granulocytic ehrlichiae warrant further investigation. We are not aware of any successful cultivation of E. phagocytophila of bovine or ovine origin in tick cells or in continuous or primary mammalian cell lines. The successful cultivation of this organism in tick cell cultures provides new opportunities for investigating several aspects of the pathogenesis and immunology of TBF in sheep and pasture fever in cattle. For example, tick cell-adapted E. phagocytophila could be used to determine whether the major surface antigens expressed in tick cells are similar to those expressed in mammalian cells (ovine and bovine granulocytes) and to investigate their use as antigens for diagnostic and prophylactic purposes.

Acknowledgments This project was supported in part by a grant from the Wellcome Trust. We are grateful to Lesley Bell-Sakyi, Centre for Tropical Veterinary Medicine, University of Edinburgh, for her invaluable advice regarding the maintenance of tick-cells in vitro, and to Nigel Jones, University of Liverpool, Department of Veterinary Clinical Science and Animal Husbandry, for providing animal care.

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References Barlough, J. E., Madigan, J. E., DeRock, E. and Bigornia, L. (1996). Nested polymerase chain reaction for detection of Ehrlichia equi genomic DNA in horses and ticks (Ixodes pacificus). Veterinary Parasitology, 63, 319±329. Byron, B. and Yanker, C. E. (1990). Improved culture conditions for Cowdria ruminantium (Rickettsiales), the agent of heartwater disease of domestic ruminants. Cytotechnology, 4, 285±290. Chen, S. M., Popov, V. L., Feng, H. M., Wen, J. and Walker, D. H. (1995). Cultivation of Ehrlichia chaffeensis in mouse embryo, Vero, BGM and L929 cells and study of Ehrlichia-induced cytopathic effect and plaque formation. Infection and Immunity, 63, 647±655. Dawson, J. E., Andersen, B. E., Fishbein, D. B., Sanchez, J. L., Goldsmith, C. S., Wilson, K. H. and Duntley, C. W. (1991). Isolation and characterisation of an Ehrlichia sp. from a patient diagnosed with human ehrlichiosis. Journal of Clinical Microbiology, 29, 2741±2745. Dawson, J. E., Candal, F. J., George, V. G. and Ades, E. W. (1993). Human endothelial cells as an alternative to DH82 cells for isolation of Ehrlichia chaffeensis, E. canis and Rickettsia rickettsii. Pathobiology, 61, 293±296. Dumler, J. S., Barbet, A. F., Bekker, C. P. J., Dasch, G. A., Palmer, G. H., Ray, S. C., Rikihisa, Y. and Rurangirwa, F. R. (2001). Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and ``HGE agent'' as synonyms of Ehrlichia phagocytophila. International Journal of Systematic Evolutionary Microbiology, 51, 2145±2165. Gocke, H. I. and Woldehiwet, Z. (1999). Lymphocyte responses to mitogens and rickettsial antigens in sheep experimentally infected with Ehrlichia (Cytoecetes) phagocytophila. Veterinary Parasitology, 83, 55±64. Goodman, J. L., Nelson, C., Vitale, B., Madigam, J. E., Dumler, J. S., Kurtti, T. J. and Munderloh, U. G. (1996). Direct cultivation of the causative agent of human granulocytic ehrlichiosis. New England Journal of Medicine, 334, 209±215. Gordon, W. S., Brownlee, A. and Wilson, D. R. (1940). Studies on louping ill, tick-borne fever and scrapie. Proceedings of the Third International Congress of Microbiology, New York, pp. 362±363. Gordon, W. S., Brownlee, A., Wilson, D. R. and MacLeod, J. (1932). Tick-borne fever (a hitherto undescribed disease of sheep). Journal of Comparative Pathology and Therapeutics, 45, 301±312. Hudson, J. R. (1950). The recognition of tick-borne fever as a disease of cattle. British Veterinary Journal, 59, 201±202. Jauron, S. D., Nelson, C. M., Fingerle, V., Ravyn, M. D., Goodman, J. I., Johnson, R. C., Lobentanzer, R., Wilske, B. and Munderloh, U. G. (2001). Journal of Infectious Diseases, 184, 1445±1450. Jongejan, F., Wassink, L. A., Thielemans, M. J. C., Perie, N. M. and Uilenberg, G. (1989). Serotypes of Cowdria ruminantium and relationship with Ehrlichia phagocytophila determined by immunofluorescence. Veterinary Microbiology, 21, 31±40.

Munderloh, U. G., Blouinm, E. F., Kocan, K. M., Ge, N. L., Edwards, W. L. and Kurtti, T. J. (1996b). Isolation and establishment of the tick (Acari: Ixodidae)-borne cattle pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) in tick cell culture. Journal of Medical Entomology, 33, 656±664. Munderloh, U. G., Jauton, S. D., Fingerle, V., Lietriz, L., Hayes, S. F., Hautman, J. M., Nelson, C. M., Huberty, B. W., Kurtti, T. J., Ahlstrand, G. G., Greig, B., Mellencamp, M. A. and Goodman, J. L. (1999). Invasion and intracellular development of the human granulocytic ehrlichiosis agent in tick cell culture. Journal of Clinical Microbiology, 37, 2518±2524. Munderloh, U. G. and Kurtti, T. J. (1989). Formulation of the medium for tick cell culture. Experimental Applied Acarology, 7, 219±229. Munderloh, U. G., Liu, Y., Wang, M., Chen, C. and Kurtti, T. (1994). Establishment, maintenance and description of cell lines from the tick Ixodes scapularis. Journal of Parasitology, 80, 533±543. Munderloh, U. G., Madigan, J. E. M., Dumler, J. S., Goodman, J. L., Hayes, S. F., Barlough, J. E., Curtis, M. N. and Kurtti, T. (1996a). Isolation of the equine granulocytic ehrlichiosis agent, Ehrlichia equi, in tick cell culture. Journal of Clinical Microbiology, 34, 664±670. Nyindo, M. B. A., Ristic, M., Huxsoll, D. L. and Smith, A. R. (1971). Tropical canine pancytopeniaÐin vitro cultivation of the causative agent, Ehrlichia canis. American Journal of Veterinary Research, 32, 1651±1658. Ogden, N., Bown, K. N. H., Horrocks, B. K., Woldehiwet, Z. and Bennett, M. (1998). Granulocytic ehrlichia infection in Ixodes ticks and mammals in woodlands and uplands of the United Kingdom. Medical and Veterinary Entomology, 12, 423±429. Richter, P. J., Kimsey, R. B., Madigan, J. E., Barlough, J. E., Dumler, J. S. and Brooks, D. L. (1996). Ixodes pacificus (Acari: Ixodidae) as a vector of Ehrlichia equi (Rickettsiales: Ehrlichieae). Journal of Medical Entomology, 33, 1±5. Telford, S. R., Dawson, J. E., Katavlos, P., Warner, C. K., Kolbert, C. P. and Persing, D. H. (1996). Perpetuation of the agent of human granulocytic ehrlichiosis in a deer tick-rodent cycle. Proceedings of the National Academy of Sciences of the USA, 93, 6209±6214. Thrusfield, M. V., Synge, B. A. and Scott, G. R. (1978). Attempts to cultivate Ehrlichia phagocytophila in vitro. Veterinary Microbiology, 2, 257±260. Tuomi, J. (1967). Experimental studies on bovine tickborne fever. (1) Clinical and haematological data, some properties of the causative agent and homologous immunity. Acta Pathologica et Microbiologica Scandinavica, 70, 577±589. Woldehiwet, Z. (1981). Observations on the immune responses of sheep infected with Cytoecetes phagocytophila. PhD Thesis, University of Edinburgh. Woldehiwet, Z. (1987). The effects of tick-borne fever on some functions of polymorphonuclear cells of sheep. Journal of Comparative Pathology, 97, 481±485. Woldehiwet, Z., Carter, S. D. and Dare, C. (1991). Purification of Cytoecetes phagocytophila, the causative agent of tick-borne fever, from infected ovine blood. Journal of Comparative Pathology, 105, 431±438.

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Woldehiwet, Z. and Scott, G. R. (1982a). Stages in the development of Cytoecetes phagocytophila, the causative agent of tick-borne fever. Journal of Comparative Pathology, 92, 469±474. Woldehiwet, Z. and Scott, G. R. (1982b). In vitro propagation of Cytoecetes phagocytophila, the causative agent of tick-borne fever. Veterinary Microbiology, 7, 127±133.

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Woldehiwet, Z. and Scott, G. R. (1993). Tick-borne (pasture) fever. In: Rickettsial and Chlamydial Diseases of Domestic Animals, Z. Woldehiwet and M. Ristic, Eds, Pergamon Press, Oxford, pp. 233±254.   Received; February 6th; 2002 Accepted; April 24th; 2002