By examination of R788 mouse IFA and ELISA, the highest titer of the polyclonal antibodies reaches 1:1600. The recombinant 56-kDa protein in the study is valuable for developing a simple and rapid diagnostic test and vaccine for O. tsutsugamushi. Scrub typhus, also known as tsutsugamushi disease, is an acute febrile illness caused by infection with O. tsutsugamushi and is characterized by fever, rash, eschar, headache and overall

soreness. The disease is endemic in the Asia–Pacific region, including China, Japan, Korea and Thailand (1–4). The incidence of the disease in humans has increased sharply in China during the past 20 years (5–7). Diagnosis of scrub typhus depends generally on clinical presentation and epidemiological history. It is very difficult to differentiate scrub typhus from other acute febrile illnesses such as murine typhus, dengue fever and viral hemorrhagic fevers because of symptom similarities (8, 9). Therefore, underdiagnosis or misdiagnosis of scrub typhus is common and may result in delayed or inappropriate treatment. Current serodiagnostic assays, such as the IFA or micro-immunofluorescent antibody assay require the propagation of Rickettsiae in infected yolk sacs of embryonated chicken eggs or antibiotic-free cell cultures

as well as special equipment such as a fluorescence microscope (10). Isolation and cultivation of O. tsutsugamushi is reliable for diagnosis but is difficult and time-consuming for a non-specialist 3-oxoacyl-(acyl-carrier-protein) reductase laboratory. PCR-based approaches that target specific O. tsutsugamushi genes also require specialist equipment (11). Therefore, a simple, rapid, BI 6727 datasheet sensitive and economic diagnostic

method, especially for use in rural areas, is urgently needed. A more practical serodiagnostic method can be developed by cloning and expressing the immunodominant genes of O. tsutsugamushi in E. coli (12–14). These recombinant proteins offer a considerable advantage over the antigen derived directly from O. tsutsugamushi because the recombinant products can be produced and purified in scalable amounts. They can then be used as antigens for developing a convenient and inexpensive diagnostic method that would greatly reduce the cost, transport expense and overcome the reproducibility problems associated with the present diagnostic tests, which require growth and purification of O. tsutsugamushi (15). Orientia tsutsugamushi is an antigenically diverse microorganism. Ohashi et al. described several antigenic variants, such as the representative strains Gilliam, Karp, Kato and other isolates (16). Most isolates of O. tsutsugamushi in China are identified as serotype Gilliam or Karp. Recent investigations suggested that the major outer membrane 56-kDa protein is a protective antigen that can be produced as a suitable recombinant protein for a diagnostic reagent purpose (15). Kim et al.