strains had varying degree of virulence, and that the antibodies in the blood sera of patients cross-react to different strains. By 1946, she established that there were three principal strains (serotypes), namely Karp (from New Guinea), Gilliam (from India) and Seerangay (from British Malaya). Akira Shishido described the Kato strain, in addition to Gilliam and Karp, in Japan in 1958. Since then, six basic antigenic strains are recognised, namely Gilliam, Karp, Kato, Shimokoshi, Kawasaki, and Kuroki. Karp is the most abundant strain, accounting for about 50% of all infections. In Korea, the major strain is Boryong. As of 2009, more than 20 different strains have been established in humans based on antigenic variation using serological tests such as complement fixation and immunofluorescence assay. The number is much higher if the strains in rodents and mites are taken into account. For example, a study in Japan in 1994 reported 32 strains, 14 from human patients, 12 from wild rodents, and 6 from trombiculid mites. The different strains exert different levels of virulence, and the most virulent is KN-3, which is predominant among wild rodents. Another study in 1996 reported 40 strains. Genetic methods have revealed even greater complexity than had been previously described (for example, Gilliam is further divided into Gilliam and JG types). Due to immunological differences of the serotypes, simultaneous and repeated infection with different strains is possible. Antigenic variation Orientia tsutsugamushi has four major surface-membrane proteins (antigens) having molecular weights 22 kDa, 47 kDa, 56 kDa and 110 kDa. A 56-kDa type specific antigen (TSA56) is the most important because it is not produced by any other bacteria, and is responsible for making the genetic diversity in different strains. It accounts for about 10–15% of the total cell proteins. The 22-kDa, 47-kDa or 110-kDa antigens are not strain specific so that TSA56 is the main target in sophisticated diagnostic tests such as immunoblotting, ELISA and DNA analysis. The protein assists the adhesion and entry of the bacterium into host cells, as well as evasion of the host's immune reaction. It varies in size from 516 to 540 amino acid residues between different strains, and its gene is approximately 1,550 base pairs long. Its gene contains four hypervariable regions, indicating that it synthesises many antigenically different proteins. There are also 11-kDa and 60-kDa proteins inside the bacterium which are very similar to GroES and GroEL of the bacterium Escherichia