Direct cultivation of the causative agent of human granulocytic ehrlichiosis

Jesse L. Goodman, Curtis Nelson, Blaise Vitale, John E Madigan, J. Stephen Dumler, Timothy J. Kurtti, Ulrike G. Munderloh

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Abstract

Abstract Background. Human granulocytic ehrlichiosis is a potentially fatal tick-borne infection that has recently been described. This acute febrile illness is characterized by myalgias, headache, thrombocytopenia, and elevated serum aminotransferase levels. The disease is difficult to diagnose because the symptoms are nonspecific, intraleukocytic inclusions (morulae) may not be seen, and the serologic results are often initially negative. Little is known about the causative agent because it has never been cultivated. Methods. We studied three patients with symptoms and laboratory findings suggestive of human granulocytic ehrlichiosis, including unexplained fever after probable exposure to ticks, granulocytopenia, and thrombocytopenia. Peripheral blood was examined for ehrlichia microscopically and with use of the polymerase chain reaction (PCR). Blood was inoculated into cultures of HL60 cells (a line of human promyelocytic leukemia cells), and the cultures were monitored for infection by Giemsa staining and PCR. Results. Blood from the three patients, only one of whom had inclusions suggestive of ehrlichia in neutrophils, was positive for human granulocytic ehrlichiosis on PCR. Blood from all three patients was inoculated into HL60 cell cultures and caused infection, with intracellular organisms visualized as early as 5 days after inoculation and cell lysis occurring within 12 to 14 days. The identity of the cultured organisms was confirmed by immunofluorescence microscopy, PCR analysis, and DNA sequencing. DNA from the infected cells was sequenced in regions of the 16S ribosomal gene reported to differ between the agent of human granulocytic ehrlichiosis and closely related species, including Ehrlichia equi and E. phagocytophila, which cause infection in animals. The sequences from all three human isolates were identical and differed from the strain of E. equi studied in having guanine rather than adenine at nucleotide 84. Conclusions. We describe the cultivation of the agent of human granulocytic ehrlichiosis in cell culture. The ability to isolate this organism should lead to a better understanding of the biology, treatment, and epidemiology of this emerging infection.

Original languageEnglish (US)
Pages (from-to)209-215
Number of pages7
JournalNew England Journal of Medicine
Volume334
Issue number4
DOIs
StatePublished - Jan 25 1996

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Ehrlichiosis
Ehrlichia
Anaplasma phagocytophilum
Polymerase Chain Reaction
Cell Culture Techniques
HL-60 Cells
Infection
Thrombocytopenia
Fever
Tick-Borne Diseases
Morula
Agranulocytosis
Adenine Nucleotides
Myalgia
Guanine
Ticks
Transaminases
DNA Sequence Analysis
Fluorescence Microscopy
Headache

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Goodman, J. L., Nelson, C., Vitale, B., Madigan, J. E., Dumler, J. S., Kurtti, T. J., & Munderloh, U. G. (1996). Direct cultivation of the causative agent of human granulocytic ehrlichiosis. New England Journal of Medicine, 334(4), 209-215. https://doi.org/10.1056/NEJM199601253340401

Direct cultivation of the causative agent of human granulocytic ehrlichiosis. / Goodman, Jesse L.; Nelson, Curtis; Vitale, Blaise; Madigan, John E; Dumler, J. Stephen; Kurtti, Timothy J.; Munderloh, Ulrike G.

In: New England Journal of Medicine, Vol. 334, No. 4, 25.01.1996, p. 209-215.

Research output: Contribution to journalArticle

Goodman, JL, Nelson, C, Vitale, B, Madigan, JE, Dumler, JS, Kurtti, TJ & Munderloh, UG 1996, 'Direct cultivation of the causative agent of human granulocytic ehrlichiosis', New England Journal of Medicine, vol. 334, no. 4, pp. 209-215. https://doi.org/10.1056/NEJM199601253340401
Goodman, Jesse L. ; Nelson, Curtis ; Vitale, Blaise ; Madigan, John E ; Dumler, J. Stephen ; Kurtti, Timothy J. ; Munderloh, Ulrike G. / Direct cultivation of the causative agent of human granulocytic ehrlichiosis. In: New England Journal of Medicine. 1996 ; Vol. 334, No. 4. pp. 209-215.
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AU - Nelson, Curtis

AU - Vitale, Blaise

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AU - Kurtti, Timothy J.

AU - Munderloh, Ulrike G.

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N2 - Abstract Background. Human granulocytic ehrlichiosis is a potentially fatal tick-borne infection that has recently been described. This acute febrile illness is characterized by myalgias, headache, thrombocytopenia, and elevated serum aminotransferase levels. The disease is difficult to diagnose because the symptoms are nonspecific, intraleukocytic inclusions (morulae) may not be seen, and the serologic results are often initially negative. Little is known about the causative agent because it has never been cultivated. Methods. We studied three patients with symptoms and laboratory findings suggestive of human granulocytic ehrlichiosis, including unexplained fever after probable exposure to ticks, granulocytopenia, and thrombocytopenia. Peripheral blood was examined for ehrlichia microscopically and with use of the polymerase chain reaction (PCR). Blood was inoculated into cultures of HL60 cells (a line of human promyelocytic leukemia cells), and the cultures were monitored for infection by Giemsa staining and PCR. Results. Blood from the three patients, only one of whom had inclusions suggestive of ehrlichia in neutrophils, was positive for human granulocytic ehrlichiosis on PCR. Blood from all three patients was inoculated into HL60 cell cultures and caused infection, with intracellular organisms visualized as early as 5 days after inoculation and cell lysis occurring within 12 to 14 days. The identity of the cultured organisms was confirmed by immunofluorescence microscopy, PCR analysis, and DNA sequencing. DNA from the infected cells was sequenced in regions of the 16S ribosomal gene reported to differ between the agent of human granulocytic ehrlichiosis and closely related species, including Ehrlichia equi and E. phagocytophila, which cause infection in animals. The sequences from all three human isolates were identical and differed from the strain of E. equi studied in having guanine rather than adenine at nucleotide 84. Conclusions. We describe the cultivation of the agent of human granulocytic ehrlichiosis in cell culture. The ability to isolate this organism should lead to a better understanding of the biology, treatment, and epidemiology of this emerging infection.

AB - Abstract Background. Human granulocytic ehrlichiosis is a potentially fatal tick-borne infection that has recently been described. This acute febrile illness is characterized by myalgias, headache, thrombocytopenia, and elevated serum aminotransferase levels. The disease is difficult to diagnose because the symptoms are nonspecific, intraleukocytic inclusions (morulae) may not be seen, and the serologic results are often initially negative. Little is known about the causative agent because it has never been cultivated. Methods. We studied three patients with symptoms and laboratory findings suggestive of human granulocytic ehrlichiosis, including unexplained fever after probable exposure to ticks, granulocytopenia, and thrombocytopenia. Peripheral blood was examined for ehrlichia microscopically and with use of the polymerase chain reaction (PCR). Blood was inoculated into cultures of HL60 cells (a line of human promyelocytic leukemia cells), and the cultures were monitored for infection by Giemsa staining and PCR. Results. Blood from the three patients, only one of whom had inclusions suggestive of ehrlichia in neutrophils, was positive for human granulocytic ehrlichiosis on PCR. Blood from all three patients was inoculated into HL60 cell cultures and caused infection, with intracellular organisms visualized as early as 5 days after inoculation and cell lysis occurring within 12 to 14 days. The identity of the cultured organisms was confirmed by immunofluorescence microscopy, PCR analysis, and DNA sequencing. DNA from the infected cells was sequenced in regions of the 16S ribosomal gene reported to differ between the agent of human granulocytic ehrlichiosis and closely related species, including Ehrlichia equi and E. phagocytophila, which cause infection in animals. The sequences from all three human isolates were identical and differed from the strain of E. equi studied in having guanine rather than adenine at nucleotide 84. Conclusions. We describe the cultivation of the agent of human granulocytic ehrlichiosis in cell culture. The ability to isolate this organism should lead to a better understanding of the biology, treatment, and epidemiology of this emerging infection.

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