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PREPARATION OF RECOMBINANT ANTIGEN OF O. TSUTSUGAMUSHI PTAN STRAIN AND DEVELOPMENT OF RAPID DIAGNOSTIC REAGENT FOR SCRUB TYPHUS

Spring scrub typhus has frequently occurred in Pingtan Island, China, since 2000. In this study, we amplified a 1352-bp DNA fragment encoding a truncated 56-kDa outer membrane protein of the Ptan strain, which was isolated from a serum sample of a patient with spring scrub typhus, and cloned it into the pET28a vector for expression. The expression product was a recombinant polypeptide containing a His-tag to facilitate purification on a Ni²⁺ chromatography column. The recombinant protein was further identified by Western blotting and enzyme-linked immunosorbent assay (ELISA) and appeared to be a good diagnostic antigen candidate. A rapid colloidal gold immunochromatographic assay (CIA) for detecting serum total antibodies, IgG and IgM, which are anti-Orientia tsutsugamushi, was developed, using a mixture of the r56 of the Gilliam and Ptan strains as the diagnostic antigen. CIA performance was tested on a panel of 112 control sera from confirmed cases of scrub typhus. The detection sensitivities of CIA against anti-O. tsutsugamushi total antibodies, IgM, and IgG were 98.2%, 81.2%, and 94.6%, respectively, while that of IFA (using the lysate of the O. tsutsugamushi Gilliam-infected chicken yolk sac as the antigen) against IgG was 85.7%. One hundred five serum samples from healthy individuals and patients with other febrile diseases were tested with CIA as negative controls. Specificities of CIA against anti-O. tsutsugamushi total antibodies, IgM, and IgG were 98.1%, 100%, and 98.9%, respectively, while the specificity of IFA against IgG was 98.9%. These results indicated that CIA was a good assay and could substitute for conventional immunofluorescence assays for diagnosis of scrub typhus.

Received December 31, 2005. Accepted for publication October 27, 2006.

Acknowledgment: We thank Dr. Qiajia Huang, Fuzhou General Hospital of Nanjing Command stationed in Fuzhou, Fujian province, China for critically reading the manuscript and helpful corrections in grammar and writing.

Financial support: This study was supported by the Bureau of Hygiene, Nanjing Command, PLA.

^* Address correspondence to Jiaqi Tang, 293 East Zhongshan Road, Nanjing, China 210002. E-mail: tjiaqi2006{at}yahoo.com.cn

Authors’ addresses: Min Cao, Hengbin Guo, Changjun Wang, Xianfu Li, Xiuzhen Pan, Zhu Jin, and Jiaqi Tang, Department of Epidemiology and Microbiology, Research Institute for Medicine of Nanjing Command, Jiangsu, China, Telephone: +86-25-84526002, Fax: +86-25-84507094, E-mail: tjiaqi2006{at}yahoo.com.cn. Tang Tang, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, E-mail: olivertt{at}gmail.com.

Min Cao, Hengbin Guo, Tang Tang, and Jiaqi Tang contributed equally to the work.