Scrub typhus (‘Tsutsugamushi’ disease in Japanese) is a mite-borne infectious disease. The causative agent is Orientia tsutsugamushi, an obligate intracellular bacterium belonging to the family Rickettsiaceae of the subdivision alpha-Proteobacteria. In this study, we determined the complete genome sequence of O. tsutsugamushi strain Ikeda, which comprises a single chromosome of 2 008 987 bp and contains 1967 protein coding sequences (CDSs). The chromosome is much larger than those of other members of Rickettsiaceae, and 46.7% of the sequence was occupied by repetitive sequences derived from an integrative and conjugative element, 10 types of transposable elements, and seven types of short repeats of unknown origins. The massive amplification and degradation of these elements have generated a huge number of repeated genes (1196 CDSs, categorized into 85 families), many of which are pseudogenes (766 CDSs), and also induced intensive genome shuffling. By comparing the gene content with those of other family members of Rickettsiacea, we identified the core gene set of the family Rickettsiaceae and found that, while much more extensive gene loss has taken place among the housekeeping genes of Orientia than those of Rickettsia, O. tsutsugamushi has acquired a large number of foreign genes. The O. tsutsugamushi genome sequence is thus a prominent example of the high plasticity of bacterial genomes, and provides the genetic basis for a better understanding of the biology of O. tsutsugamushi and the pathogenesis of ‘Tsutsugamushi’ disease.

Recent studies of Rickettsia tsutsugamushi have demonstrated clearly the phenotypic and genotypic differences between this microorganism and other species belonging to the genus Rickettsia. Therefore, classification of R. tsutsugamushi in a new genus, Orientiu gen. nov., is proposed. The genus Rickettsia includes etiological agents of human diseases, including typhus, spotted fever, and scrub typhus. All of the microbes belonging to this genus are similar in the following respects: they exhibit obligate intracellular parasitism, they are morphologically similar to gram-negative bacteria, they survive in both vertebrate and arthropod hosts, and human infection is mediated by arthropods. However, recent studies of Rickettsia tsutsugamushi, which is the only species in the scrub typhus group, have revealed some differences between this species and other species belonging to the genus Rickettsia. The most striking difference is in the structure of the outer envelope; as revealed by electron microscopy, the outer leaflet of the cell wall of R. tsutsugamushi is considerably thicker than the inner leaflet, while the opposite is true of the other Rickettsia species (36). In addition, chemically, R. tsutsugamushi lacks constitutional components of peptidoglycan and lipopolysaccharide, such as muramic acid, glucosamine, hydroxy fatty acids, and 2-keto-3-deoxyoctonic acid, suggesting that neither peptidoglycan nor lipopolysaccharide is present in R tsutsugamushi (4), while these substances are generally found in the other species belonging to the genus Rickettsia (1, 8,15, 24,35). R. tsutsugamushi is very soft and fragile (39, 44), which reflects the lack of peptidoglycan in this microorganism, and the growth of R tsutsugamushi is more resistant to penicillin than the growth of other rickettsiae (21, 32). The protein composition of R. tsutsugamushi as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is very different from the protein compositions of the other rickettsiae, especially in the envelopes (43). In R. tsutsugamushi, a 54-to 58-kDa protein (designated the 56-kDa protein) is most abundant and is located on the cell surface (16, 41). Other major proteins (80, 46, 43,39, 35,28, and 25 kDa) are also located on the rickettsial surface (41); three of these proteins, the 25-,28-, and also 56-kDa proteins, are heat modifiable (16, 22, 46). Another abundant protein, the 60-kDa protein, which is located inside the rickettsial cells, has been shown to exhibit homology to the GroEL family of proteins (37). On the other hand, rickettsiae belonging to the typhus and spotted fever groups resemble each other in their protein compositions. The major antigenic proteins located on the surfaces of these organisms have been estimated to have the molecular sizes of 150 to 180, 110 to 130,49,32,27.5, and 16.5 to 17.5 kDa (51,53). The two proteins larger than 110 kDa are immunodominant antigens and have both heat-labile and heatstable antigenic sites (5-7, 12, 15). The 16.5-to 17.5-kDa antigen is a lipoprotein and e...

Polyacrylamide gel electrophoresis of lysates of purified Rickettsia tsutsugamushi revealed as many as 30 polypeptide bands, including major bands corresponding to molecular sizes of 70, 60, 54 to 56, and 46 to 47 kilodaltons. Compared with the polypeptide composition of the rickettsiae of Gifliam, Karp, and Kato strains and a newly isolated Shimokoshi strain, the major polypeptide in the Kato strain (54-56K) and in the Karp strain (46-47K) migrated a little faster and slower, respectively, than the corresponding polypeptides in the other strains. The largest major polypeptide (54-56K) was digestible by the treatment of intact rickettsiae with trypsin and variable in content in separate preparations, suggesting that the polypeptide exists on the rickettsial surface and is easily degraded during the handling of these microorganisms. Several surface polypeptides of rickettsiae, including the 54-56K and 46-47K polypeptides, were detected by radioiodination of intact rickettsiae foliowed by polyacrylamide gel electrophoresis of the lysate; however, the 70K and 60K polypeptides were not labeled. Immunoblotting experiments with hyperimmune sera prepared in guinea pigs against each strain demonstrated that the 70K, 54-56K, and 46-47K polypeptides showed antigenic activities. The 54-56K polypeptide appeared to be strain specific, whereas the 70K and 46-47K polypeptides cross-reacted with the heterologous antisera.on July 15, 2020 by guest http://iai.asm.org/ Downloaded from

A total of 40 strains of Orientia tsutsugamushi (34 isolates from patients and trombiculid mites in Japan, and 6 prototype strains of antigenic variants) were examined for classification based on the reactivities with type-specific monoclonal antibodies in indirect immunofluorescence tests, and on the restriction fragment length polymorphism of a polymerase chain reaction (PCR)-amplified 56-kilodalton type-specific antigenic protein gene. By these methods, several antigenic and genotypic variants were found among the strains, and these variants were classified into types and further into subtypes. These results suggest that there are many variants in O. tsutsugamushi, and the methods used here seem to be useful for the systematic classification of the numerous variants. A strain which may be a new type distinguishable from those identified previously was also found in this study. Furthermore, variety in the degree of pathogenicity in mice related to type and/or subtype classification were observed.

Summaryobjectives To identify different rickettsial infections using a specific immunofluorescent technique in patients clinically diagnosed as 'typhus fever' in the Central Province of Sri Lanka, and to define the clinical picture, assess the severity of infection and to determine the pattern of geographical distribution of the infections of the hospital-based patients.methods A specific indirect immunofluorescent antibody technique was used on the sera of two groups of patients in laboratories in Japan and Thailand.results We serodiagnosed infections with Orientia tsutsugamushi, Rickettsia typhi and spotted fever group in 56 of 118 clinically investigated patients. There were eight infections with O. tsutsugamushi, two with R. typhi and 10 spotted fever group patients with IgM antibodies suggestive of acute infection. Nineteen patients had antibodies against these three rickettsial species, suggestive of past exposure, co-infection or cross-reactivity of antigens. Discrete, erythematous maculopapular rash was common to all three types of infection except for five patients who had no rash. Five patients positive for spotted fever antibodies developed fern-leaf type skin necrosis with severe illness. Duration of the febrile period ranged from 4 to 23 days with defervescence occurring after specific antibiotic treatment.conclusions The study has shown the presence of different types of rickettsial infections in the Central Province of Sri Lanka. The characterization of the clinical picture and the severity of infection provide useful information for the proper management of the patients in the future.