Munderloh U G, Madigan J E, Dumler J S, Goodman J L, Hayes S F, Barlough J E, Nelson C M, Kurtti T J
Department of Entomology, University of Minnesota, St. Paul 55108, USA.
J Clin Microbiol. 1996 Mar;34(3):664-70. doi: 10.1128/jcm.34.3.664-670.1996.
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.
马粒细胞埃立克体病病原体——马埃立克体，与人粒细胞埃立克体病（HGE）病原体密切相关或完全相同。二者均怀疑由蜱传播。我们已成功地在一种源自假定传播媒介肩突硬蜱的细胞系IDE8中分离出了马埃立克体。将实验感染马的外周血白细胞接种到IDE8单层细胞上。培养物在含有NaHCO₃和有机缓冲液[3 -（N - 吗啉代）- 丙烷磺酸]（MOPS）的蜱细胞培养基中，于34℃的烛缸内培养。两周内，在吉姆萨染色的培养样本中检测到了受感染细胞，随后这些病原体扩散到培养物中的未感染细胞。每隔2至3周，通过将一部分受感染培养物转移到新的细胞层，马埃立克体得以连续传代。使用针对马埃立克体和HGE病原体的寡核苷酸引物进行PCR以及通过免疫细胞学法确认了这些病原体的身份。同源马抗体和人抗HGE恢复期血清识别在蜱细胞培养物中生长的马埃立克体。电子显微镜显示，在宿主细胞内体中形成了电子透明和致密的类埃立克体形态。在蜱细胞培养物中传代两次的马埃立克体对马宿主仍保持感染力和致病性，静脉注射感染蜱细胞悬液并随后从外周血中重新分离得到病原体，从而满足了科赫法则。该马在1周内出现了严重的临床症状，即发热、食欲不振、血小板减少、黄疸和肢体水肿，这些都是粒细胞性马埃立克体病的典型症状。
