Skip to main content
NCBI home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1996 Mar;34(3):664–670. doi: 10.1128/jcm.34.3.664-670.1996

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

U G Munderloh 1, J E Madigan 1, J S Dumler 1, J L Goodman 1, S F Hayes 1, J E Barlough 1, C M Nelson 1, T J Kurtti 1
PMCID: PMC228866  PMID: 8904434

Abstract

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.

Full Text

The Full Text of this article is available as a PDF (2.1 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson B. E., Dawson J. E., Jones D. C., Wilson K. H. Ehrlichia chaffeensis, a new species associated with human ehrlichiosis. J Clin Microbiol. 1991 Dec;29(12):2838–2842. doi: 10.1128/jcm.29.12.2838-2842.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bakken J. S., Dumler J. S., Chen S. M., Eckman M. R., Van Etta L. L., Walker D. H. Human granulocytic ehrlichiosis in the upper Midwest United States. A new species emerging? JAMA. 1994 Jul 20;272(3):212–218. [PubMed] [Google Scholar]
  3. Brenner D. J., O'Connor S. P., Winkler H. H., Steigerwalt A. G. Proposals to unify the genera Bartonella and Rochalimaea, with descriptions of Bartonella quintana comb. nov., Bartonella vinsonii comb. nov., Bartonella henselae comb. nov., and Bartonella elizabethae comb. nov., and to remove the family Bartonellaceae from the order Rickettsiales. Int J Syst Bacteriol. 1993 Oct;43(4):777–786. doi: 10.1099/00207713-43-4-777. [DOI] [PubMed] [Google Scholar]
  4. Brouqui P., Dumler J. S., Lienhard R., Brossard M., Raoult D. Human granulocytic ehrlichiosis in Europe. Lancet. 1995 Sep 16;346(8977):782–783. doi: 10.1016/s0140-6736(95)91544-3. [DOI] [PubMed] [Google Scholar]
  5. Burgdorfer W., Barbour A. G., Hayes S. F., Benach J. L., Grunwaldt E., Davis J. P. Lyme disease-a tick-borne spirochetosis? Science. 1982 Jun 18;216(4552):1317–1319. doi: 10.1126/science.7043737. [DOI] [PubMed] [Google Scholar]
  6. Chen S. M., Dumler J. S., Bakken J. S., Walker D. H. Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease. J Clin Microbiol. 1994 Mar;32(3):589–595. doi: 10.1128/jcm.32.3.589-595.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dawson J. E., Anderson B. E., Fishbein D. B., Sanchez J. L., Goldsmith C. S., Wilson K. H., Duntley C. W. Isolation and characterization of an Ehrlichia sp. from a patient diagnosed with human ehrlichiosis. J Clin Microbiol. 1991 Dec;29(12):2741–2745. doi: 10.1128/jcm.29.12.2741-2745.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dumler J. S., Asanovich K. M., Bakken J. S., Richter P., Kimsey R., Madigan J. E. Serologic cross-reactions among Ehrlichia equi, Ehrlichia phagocytophila, and human granulocytic Ehrlichia. J Clin Microbiol. 1995 May;33(5):1098–1103. doi: 10.1128/jcm.33.5.1098-1103.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dumler J. S., Chen S. M., Asanovich K., Trigiani E., Popov V. L., Walker D. H. Isolation and characterization of a new strain of Ehrlichia chaffeensis from a patient with nearly fatal monocytic ehrlichiosis. J Clin Microbiol. 1995 Jul;33(7):1704–1711. doi: 10.1128/jcm.33.7.1704-1711.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. FOGGIE A. Studies on the infectious agent of tick-borne fever in sheep. J Pathol Bacteriol. 1951 Jan;63(1):1–15. doi: 10.1002/path.1700630103. [DOI] [PubMed] [Google Scholar]
  11. Ge N. L., Kocan K. M., Murphy G. L., Blouin E. F. Detection of Anaplasma marginale DNA in bovine erythrocytes by slot-blot and in situ hybridization with a PCR-mediated digoxigenin-labeled DNA probe. J Vet Diagn Invest. 1995 Oct;7(4):465–472. doi: 10.1177/104063879500700407. [DOI] [PubMed] [Google Scholar]
  12. Goodman J. L., Nelson C., Vitale B., Madigan J. E., Dumler J. S., Kurtti T. J., Munderloh U. G. Direct cultivation of the causative agent of human granulocytic ehrlichiosis. N Engl J Med. 1996 Jan 25;334(4):209–215. doi: 10.1056/NEJM199601253340401. [DOI] [PubMed] [Google Scholar]
  13. Herwaldt B. L., Springs F. E., Roberts P. P., Eberhard M. L., Case K., Persing D. H., Agger W. A. Babesiosis in Wisconsin: a potentially fatal disease. Am J Trop Med Hyg. 1995 Aug;53(2):146–151. doi: 10.4269/ajtmh.1995.53.146. [DOI] [PubMed] [Google Scholar]
  14. Hitchcock P. J., Brown T. M., Corwin D., Hayes S. F., Olszewski A., Todd W. J. Morphology of three strains of contagious equine metritis organism. Infect Immun. 1985 Apr;48(1):94–108. doi: 10.1128/iai.48.1.94-108.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ito S., Vinson J. W., McGuire T. J., Jr Murine typhus Rickettsiae in the Oriental rat flea. Ann N Y Acad Sci. 1975;266:35–60. doi: 10.1111/j.1749-6632.1975.tb35087.x. [DOI] [PubMed] [Google Scholar]
  16. Johansson K. E., Pettersson B., Uhlén M., Gunnarsson A., Malmqvist M., Olsson E. Identification of the causative agent of granulocytic ehrlichiosis in Swedish dogs and horses by direct solid phase sequencing of PCR products from the 16S rRNA gene. Res Vet Sci. 1995 Mar;58(2):109–112. doi: 10.1016/0034-5288(95)90061-6. [DOI] [PubMed] [Google Scholar]
  17. Lewis G. E., Jr, Huxsoll D. L., Ristic M., Johnson A. J. Experimentally induced infection of dogs, cats, and nonhuman primates with Ehrlichia equi, etiologic agent of equine ehrlichiosis. Am J Vet Res. 1975 Jan;36(1):85–88. [PubMed] [Google Scholar]
  18. Madigan J. E., Barlough J. E., Dumler J. S., Schankman N. S., DeRock E. Equine granulocytic ehrlichiosis in Connecticut caused by an agent resembling the human granulocytotropic ehrlichia. J Clin Microbiol. 1996 Feb;34(2):434–435. doi: 10.1128/jcm.34.2.434-435.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Madigan J. E., Gribble D. Equine ehrlichiosis in northern California: 49 cases (1968-1981). J Am Vet Med Assoc. 1987 Feb 15;190(4):445–448. [PubMed] [Google Scholar]
  20. Madigan J. E., Hietala S., Chalmers S., DeRock E. Seroepidemiologic survey of antibodies to Ehrlichia equi in horses of northern California. J Am Vet Med Assoc. 1990 Jun 15;196(12):1962–1964. [PubMed] [Google Scholar]
  21. Madigan J. E., Richter P. J., Jr, Kimsey R. B., Barlough J. E., Bakken J. S., Dumler J. S. Transmission and passage in horses of the agent of human granulocytic ehrlichiosis. J Infect Dis. 1995 Oct;172(4):1141–1144. doi: 10.1093/infdis/172.4.1141. [DOI] [PubMed] [Google Scholar]
  22. Magnarelli L. A., Dumler J. S., Anderson J. F., Johnson R. C., Fikrig E. Coexistence of antibodies to tick-borne pathogens of babesiosis, ehrlichiosis, and Lyme borreliosis in human sera. J Clin Microbiol. 1995 Nov;33(11):3054–3057. doi: 10.1128/jcm.33.11.3054-3057.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Magnarelli L. A., Stafford K. C., 3rd, Mather T. N., Yeh M. T., Horn K. D., Dumler J. S. Hemocytic rickettsia-like organisms in ticks: serologic reactivity with antisera to Ehrlichiae and detection of DNA of agent of human granulocytic ehrlichiosis by PCR. J Clin Microbiol. 1995 Oct;33(10):2710–2714. doi: 10.1128/jcm.33.10.2710-2714.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Munderloh U. G., Kurtti T. J. Formulation of medium for tick cell culture. Exp Appl Acarol. 1989 Aug;7(3):219–229. doi: 10.1007/BF01194061. [DOI] [PubMed] [Google Scholar]
  25. Munderloh U. G., Liu Y., Wang M., Chen C., Kurtti T. J. Establishment, maintenance and description of cell lines from the tick Ixodes scapularis. J Parasitol. 1994 Aug;80(4):533–543. [PubMed] [Google Scholar]
  26. Pancholi P., Kolbert C. P., Mitchell P. D., Reed K. D., Jr, Dumler J. S., Bakken J. S., Telford S. R., 3rd, Persing D. H. Ixodes dammini as a potential vector of human granulocytic ehrlichiosis. J Infect Dis. 1995 Oct;172(4):1007–1012. doi: 10.1093/infdis/172.4.1007. [DOI] [PubMed] [Google Scholar]
  27. Piesman J., Mather T. N., Telford S. R., 3rd, Spielman A. Concurrent Borrelia burgdorferi and Babesia microti infection in nymphal Ixodes dammini. J Clin Microbiol. 1986 Sep;24(3):446–447. doi: 10.1128/jcm.24.3.446-447.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. REYNOLDS E. S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol. 1963 Apr;17:208–212. doi: 10.1083/jcb.17.1.208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rikihisa Y. The tribe Ehrlichieae and ehrlichial diseases. Clin Microbiol Rev. 1991 Jul;4(3):286–308. doi: 10.1128/cmr.4.3.286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rodgers S. J., Morton R. J., Baldwin C. A. A serological survey of Ehrlichia canis, Ehrlichia equi, Rickettsia rickettsii, and Borrelia burgdorferi in dogs in Oklahoma. J Vet Diagn Invest. 1989 Apr;1(2):154–159. doi: 10.1177/104063878900100212. [DOI] [PubMed] [Google Scholar]
  31. Sato T. A modified method for lead staining of thin sections. J Electron Microsc (Tokyo) 1968;17(2):158–159. [PubMed] [Google Scholar]
  32. Schachter J., Caldwell H. D. Chlamydiae. Annu Rev Microbiol. 1980;34:285–309. doi: 10.1146/annurev.mi.34.100180.001441. [DOI] [PubMed] [Google Scholar]
  33. Sitbon M., Sola B., Evans L., Nishio J., Hayes S. F., Nathanson K., Garon C. F., Chesebro B. Hemolytic anemia and erythroleukemia, two distinct pathogenic effects of Friend MuLV: mapping of the effects to different regions of the viral genome. Cell. 1986 Dec 26;47(6):851–859. doi: 10.1016/0092-8674(86)90800-7. [DOI] [PubMed] [Google Scholar]
  34. Smith R. D., Sells D. M., Stephenson E. H., Ristic M. R., Huxsoll D. L. Development of Ehrlichia canis, causative agent of canine ehrlichiosis, in the tick Rhipicephalus sanguineus and its differentiation from a symbiotic Rickettsia. Am J Vet Res. 1976 Feb;37(2):119–126. [PubMed] [Google Scholar]
  35. Stannard A. A., Gribble D. H., Smith R. S. Equine ehrlichiosis: a disease with similarities to tick-borne fever and bovine petechial fever. Vet Rec. 1969 Feb 8;84(6):149–150. doi: 10.1136/vr.84.6.149. [DOI] [PubMed] [Google Scholar]
  36. Winjum N., Riley L. K. In vitro proliferation of a canine granulocytic Ehrlichia. Vet Microbiol. 1993 Apr;34(4):355–362. doi: 10.1016/0378-1135(93)90060-k. [DOI] [PubMed] [Google Scholar]
  37. Woldehiwet Z., Scott G. R. In vitro propagation of Cytoecetes phagocytophila, the causative agent of tick-borne fever. Vet Microbiol. 1982 May;7(2):127–133. doi: 10.1016/0378-1135(82)90024-4. [DOI] [PubMed] [Google Scholar]
  38. Woldehiwet Z., Scott G. R. Stages in the development of Cytoecetes phagocytophila, the causative agent of tick-borne fever. J Comp Pathol. 1982 Jul;92(3):469–474. doi: 10.1016/0021-9975(82)90033-0. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES