Hiroshi Urakami

Classification of Rickettsia tsutsugamushi in a new genus, Orientia gen. nov., as Orientia tsutsugamushi comb. nov.

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, Orientia gen. nov., is proposed.

The Whole-genome Sequencing of the Obligate Intracellular Bacterium Orientia tsutsugamushi Revealed Massive Gene Amplification During Reductive Genome Evolution

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.

Purification of Rickettsia tsutsugamushi by Percoll Density Gradient Centrifugation

Purification of Rickettsia tsutsugamushi has been achieved by Percoll density gradient centrifugation. The microorganisms purified showed good retention of infectivity and intracellular morphology. Budding rickettsiae in the egressing stage and intracellular rickettsiae in the multiplying process were harvested separately and purified by this technique. In electron microscopic observations, the intracellular rickettsiae obtained were surrounded with double membrane‐layers of cell wall and cell membrane, and the budding rickettsiae were enveloped with an additional outermost membrane which may have originated from host cell membrane obtained in the budding process.

Demonstration of Antigenic and Genotypic Variation in Orientia tsutsugamushi Which Were Isolated in Japan, and Their Classification into Type and Subtype

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.

Isolation of Rickettsia tsutsugamushi Antigenically Different from Kato, Karp, and Gilliam Strains from Patients

Rickettsia tsutsugamushi strains from three recent patients of Tsutsugamushi disease in Niigata Prefecture were isolated primarily in mice and then in L cell cultures. By this procedure, low virulent strains to mice, as well as high virulent ones, could be isolated and cultivated serially in L cell cultures, suggesting the usefulness of L cells for isolation of this species of rickettsia. Each newly isolated strain was identified as a member of R. tsutsugamushi from the results of cross immunological tests and morphological observation. On the other hand, it was recognized that one of these rickettsiae showed immunological properties distinguishable from the prototype strains of Kato, Karp, and Gilliam by the cross complement fixation test, and also had low virulence in mice.

Penetration of Rickettsia tsutsugamushi into cultured mouse fibroblasts (L cells): an electron microscopic observation.

The mechanism of penetration of purified Rickettsia tsutsugamushi (Gilliam strain) into cultured mouse fibroblasts (L cells) was examined by electron microscopy. After 10–40 min of infection, rickettsiae in the process of being phagocytized were often seen on the cell surface. These were restricted to the rickettsiae which seemed to be intact in morphology, while heavily plasmolyzed ones were never phagocytized. Additionally, rickettsiae were taken up individually into a phagosome, and phagocytosis of several rickettsiae together was rarely observed, except in the case of heat‐inactivated microorganisms. In the cells, phagosomes whose membranes enclosed rickettsiae either tightly or loosely were seen. Rickettsiae in the loose phagosomes often showed signs of plasmolysis and were rarely released into the cell cytoplasm. Partial disintegration of phagosomal membranes and the escape of rickettsiae from the phagosomes were seen only in tight phagosomes. Large phagosomes containing a clump of several rickettsiae were observed occasionally, in which case the microorganisms were deformed and seemed to be denatured. From the above observations and the frequency of appearance of these different penetration stages in the specimens 10, 20, and 40 min after infection, it was concluded that the rickettsiae enter initially into a tight phagosome by phagocytosis and are then released into the cell cytoplasm by disruption of the phagosomal membrane. No other mechanisms of penetration were found. On the other hand, rickettsiae inactivated by trypsin did not attach to host cells. Inactivation by heat or UV irradiation resulted in reduction of phagocytosis, and rickettsiae treated with rifamycin could penetrate into the host cell cytoplasm to the same extent as in the case of infection with intact rickettsiae.

Chlorine Sensitivity of Feline Calicivirus, a Norovirus Surrogate

ABSTRACT The sensitivity to free chlorine of feline calicivirus (FCV), a norovirus surrogate, was examined relative to chlorine demand. When a crude suspension of FCV was treated with a sodium hypochlorite solution containing 10 μg/ml free chlorine, the extent of the decrease of viral infectivity clearly depended on the volume of the reaction mixture. The apparent sensitivity of FCV to free chlorine increased with the reduction of host cell debris, indicating that chlorine demand must be minimized to know the true sensitivity of the virus. We therefore partially purified the viruses from the host cell components and found that the infectivity of FCV was reduced by more than log 4.6 by 5 min of treatment with 300 ng/ml free chlorine.

Electron Microscopic Studies on Intracellular Multiplication of Rickettsia tsutsugamushi in L Cells

The mechanism and kinetics of intracellular growth of Rickettsia tsutsugamushi were investigated by electron microscopic observations, parallel with quantitative analysis by counting the rickettsiae seen in electron micrographs and by plaque assay for infectivity of the culture. The observations demonstrated the existence of electron‐less dense and ‐dense types of rickettsiae in the early stage of infection, binary fission and the process of release of the microorganisms in the host cell cytoplasm and from the cell surface, formation of abnormally long rickettsiae, and the process of lysis of the host cell in the later stage of infection with vacuole formation between the inner and outer leaflets of the host cell nuclear membrane. Separate titrations of infectivity of the cells and the culture fluid showed a very slow increase in infectivity in the culture fluid compared with the intracellular titer, suggesting that the progeny rickettsiae stay in the cell or at the cell surface for a relatively long period. Doubling time of the rickettsia was found to be about 9 hr.

Genome Comparison and Phylogenetic Analysis of Orientia tsutsugamushi Strains

Orientia tsutsugamushi (OT) is an obligate intracellular bacterium belonging to the family Rickettsiaceae and is the causative agent of scrub typhus, or Tsutsugamushi disease. The complete genome sequences of two OT strains (Boryong and Ikeda) have recently been determined. In the present study, we performed a fine genome sequence comparison of these strains. Our results indicate that although the core gene set of the family Rickettsiaceae is highly conserved between the two strains, a common set of repetitive sequences have been explosively amplified in both genomes. These amplified repetitive sequences have induced extensive genome shuffling and duplications and deletions of many genes. On the basis of the results of the genome sequence comparison, we selected 11 housekeeping genes and carried out multilocus sequence analysis of OT strains using the nucleotide sequences of these genes. This analysis revealed for the first time the phylogenetic relationships of representative OT strains. Furthermore, the results suggest the presence of an OT lineage with higher potential for virulence, which may explain the clinical and epidemiological differences between ‘classic’ and ‘new’ types of Tsutsugamushi disease in Japan.

Phylogenetic characterization of Orientia tsutsugamushi isolated in Taiwan according to the sequence homologies of 56-kDa type-specific antigen genes.

Fourteen strains of Orientia tsutsugamushi isolated in Taiwan were characterized by sequencing 56‐kDa type‐specific antigen genes and patterns of restriction fragment length polymorphism (RFLP) predicted by a computer program. The strains showed high varieties in sequence homologies and were classified to 10 types by predicted patterns of RFLP. Furthermore, all Taiwan strains were not identical in typing with strains analyzed previously. These results suggest that there are various types of O. tsutsugamushi in Taiwan that are different from those distributed in other countries.