恙虫病东方体的分离及外膜蛋白基因序列分析

Isolation of oriential tsutsugamushi and sequence analysis on it's 56kD protein gene of scrub typhus

目的 对福建省平潭岛的恙虫病病人、宿主动物和媒介恙螨进行现场调查和恙虫病东方体分离 ,并对其5 6kD外膜蛋白基因序列进行测定和分析 ,为有针对性的防制提供依据。方法 将病人血、鼠脏器、地里纤恙螨标本分别腹腔接种小白鼠分离东方体 ,小鼠腹壁刮液经Gimesa染色镜检 ;PCR扩增分离株的东方体 5 6kD外膜蛋白基因片段 ,并进行测序及多序列比较和进化树分析。结果 其中 1份病人血标本、1份鼠脏器标本及 1份恙螨标本腹腔接种的小白鼠 ,12d后出现耸毛、弓背、厌食、腹部增大等典型东方体感染症状 ,解剖后见肝脾肿大或腹水 ,显微镜下清淅可见细胞浆内短杆状或球杆状东方体 ,共分离出Pt1、Pt2、及Pt33株东方体。 3株东方体用设计的引物分别扩增出约 135 0bp特异性 5 6kD基因片段。序列测定结果表明 ,此 3株东方体之间 5 6kD基因片段同源性大于 99%。它们与Ot标准株Karp、Kato、Gilliam及地方株Kawassaki、Kuroki的同源性分别为 93 8% ,6 6 2 % ,85 1% ,6 9 6 % ,84 2 %。 结论 从恙虫病病人、宿主及媒介均分离到恙虫病东方体 ,证实了平潭岛恙虫病春季恙虫病的存在 。

Objective To isolate orientia Tsutsugamushi (Ot) from patients,host animal(Rodents) and vector mite(trombiculid mite) on Pingtan island and analyze the gene of Ot DNA for the purpose of prevention.Methods The mice were peritoneal injected by untreated patient's blood,spleen of rodent and leptotrombidium(L.) Deliense respectively and the Ot were observed from infected mice on the microscope.Then the 1?350?bp DNA fragment encoding the Ot 56kD protein can be amplified after Ot DNA being extracted from spleen of the infected mice.The specific genes were sequenced and compared with those of standard strains karp,kato,Gilliam,kawasaki and Kuroki for their homology analysis.Results Three Ot strains were isolated from patient,host rodent and vector mite respectively.The typical Ot can be seen in the cytoplasm of infected cell on the microscope.About 1?350?bp DNA fragments of 56kD protein were amplified and sequenced.The three isolates were almost identical to each other (with nucleotide sequence homology >99 5%).Compared with Karp,kato,Gilliam,Kawasaki,Kuroki,their gene sequence homology of 56kD protein were 93 8%,66 2%,85 1%,69 6%,84 2% respectively.Conclusion This was the first report of scrub typhus in the season of spring and also firstly identifying the Ot by molecular technique on Pingtan island.The Ot strains here were highly homologous to Karp type.