Abstract: Objective To isolate and identify Orientia tsutsugamushi from rodents in Dali city, Yunnan province, China, and to amplify and analyze its full-length 56 kDa type-specific antigen (TSA) gene. Methods From August 2020 to August 2021, rodents were collected from four townships in Dali city using the night trapping method. Nested polymerase chain reaction (PCR) was used to amplify the conserved sequence of O. tsutsugamushi. PCR-positive samples were taken to intraperitoneally inoculate mice to isolate O. tsutsugamushi. The isolated O. tsutsugamushi was identified by observation with Giemsa staining and indirect immunoassay. Primers were designed to amplify the full-length 56 kDa TSA gene of O. tsutsugamushi. Bioinformatics software (MEGA Ⅹ, DNAStar 7.1, and SeqMan) was used for homology and phylogenetic analyses. Results A total of 76 rodents were captured, including 6 species under 5 genera. PCR detected the sequence of O. tsutsugamushi from the liver and spleen tissue homogenate of a Rattus tanezumi, with a positive rate of 2.38% (1/42). One strain named DALIV8 was isolated and identified as O. tsutsugamushi. The full-length 56 kDa TSA gene of this isolate was 1 566 bp, encoding 521 amino acids (GenBank:OM914742). The 56 kDa TSA gene of the DALIV8 strain showed similarities of 82.30%-98.85% at the nucleotide level and 74.04%-98.28% at the amino acid level to the Kawasaki, Boryong, Karp, Kato, and TA763 genotypes. So, the isolate was identified as the TA763 genotype. Conclusion The DALIV8 strain isolated from R. tanezumi in Dali city is firstly recorded as the TA763 genotype of O. tsutsugamushi in Yunnan province, China.

Key words: Orientia tsutsugamushi, Isolation, Identification, Molecular characteristic, Dali

摘要： 目的 从云南省大理市啮齿动物中分离和鉴定恙虫病东方体（Orientia tsutsugamsushi，Ot），并对分离到的Ot 56 kDa型特异性抗原（56 kDa TSA）基因全长进行扩增分析。方法 2020年8月－2021年8月采用夹夜法，在大理市4个乡（镇）采集啮齿动物样本，巢氏PCR扩增Ot保守基因，阳性样品采用小鼠腹腔接种法分离Ot。分离到的Ot用吉姆萨染色观察和间接免疫荧光试验进行鉴定，设计引物扩增Ot的56 kDa TSA全长，用MEGA Ⅹ、DNAStar 7.1、SeqMan等生物信息学软件进行同源性与遗传进化分析。结果 共捕获啮齿动物5属6种76只，从1只黄胸鼠肝、脾组织研磨液中扩增出了Ot序列，阳性率为2.38%（1/42）。分离和鉴定了1株Ot，命名为DALIV8。该分离株56 kDa TSA基因全长1 566 bp，编码521个氨基酸（GenBank：OM914742）。DALIV8株56 kDa TSA基因在核苷酸水平与Kawasaki、Boryong、Karp、Kato、TA763基因型相似性为82.30%～98.85%，在氨基酸水平相似性为74.04%～98.28%。该Ot分离株为TA763基因型。结论 云南省大理市黄胸鼠Ot分离株DALIV8，经对其56 kDa TSA基因的分子鉴定，确定为云南省首次分离获得Ot TA763基因型。

关键词: 恙虫病东方体, 分离, 鉴定, 基因特征, 大理