Electron-microscopic observation of mouse spleen tissue infected with Orientia tsutsugamushi isolated from Shandong, China.

Low-virulent Orientia tsutsugamushi (Ot) were successfully isolated from scrub typhus patients in Shandong, China, and the isolates were similar to the Kawasaki type identified by nested polymerase chain reaction (PCR). To identify the morphological characterization of the low-virulent Ot, and elucidate the pathological changes on host cells, mouse spleen tissue infected with the Ot isolated from Shandong was used for the ultrastructural study. Transmission electron microscopy showed that the Ot parasitized in the spleen were different in size, shape and electron density and many significant changes occurred in cytoplasmic organelles of the inoculated mouse spleen cells. Swollen perinuclear cisterna was observed in the nuclear membranes of mononuclear cells and a multivesicular body was found in the intracytoplasm of the macrophage. In the phagosome of the macrophage, many Ot enveloped with an additional membrane were found to push the phagosomal membrane outward from inside. The results indicated that the low-virulent Ot and the spleen cells suffered various damages.

Ultrastructural visualization of Orientia tsutsugamushi in biopsied eschars and monocytes from scrub typhus patients in South Korea.

Scrub typhus, which is caused by Orientia tsutsugamushi, is a public health problem in the Asian-Pacific region and is the third most frequently reported infectious disease in South Korea. While ultrastructural studies have been performed on O. tsutsugamushi in murine fibroblasts, its variable locations in patients have hampered similar studies in humans. Two patients with scrub typhus agreed to provide an eschar biopsy and peripheral blood, respectively. Transmission electron microscopy was performed separately on the necrotic crust and perifocal skin of the eschar, the peripheral blood, and the infected murine L cells. O. tsutsugamushi was located within or adjacent to the outermost layer of the perifocal inflamed skin of the eschar but not in the necrotic centre. O. tsutsugamushi in peripheral blood monocytes exhibited the characteristic features of O. tsutsugamushi in L cells, namely, nearly round shaped bacteria with a size of 1-2 µm and a double membrane bearing a clear halo-like outer layer. The findings confirmed that the bacterium was predominantly located in the inflamed skin around the eschar and that the bacterium had the same ultrastructural features in human monocytes as in L cells. These findings suggest that the perifocal area, not the necrotic centre, should be sampled for diagnosis.

Orientia tsutsugamushi infection: overview and immune responses.

Orientia tsutsugamushi, an obligate intracellular bacterium, was isolated for the first time in 1930. Infections by virulent strains are characterized by fever, rash, eschar, pneumonia, myocarditis, and disseminated intravascular coagulation. Here we review the general aspects of O. tsutsugamushi and immune responses in terms of inflammation, protective immune mechanisms, and immunogenic antigens.

An ultrastructural study of vertical transmission of Rickettsia tsutsugamushi during oogenesis and spermatogenesis in Leptotrombidium pallidum.

Rickettsia tsutsugamushi in Leptotromibidium pallidum was observed by electron microscopy and rickettsiae were found in the various tissues and organs of both larvae and adults. Budding of rickettsiae, a manner of release from the host cells, was observed only in the rudiments of the reproductive organs in larvae. Oogonia and maturing oocytes in adult females and eggs after oviposition contained the microorganisms. In adult males, rickettsiae were also found in the spermatogonia, spermatocytes, and spermatids in the early stage of spermatogenesis, but were eliminated from these cells during maturation. Only the maturing spermatids, but not the eliminated rickettsiae, migrated to another rickettsia-free area of the testis, resulting in the separation of spermatids from rickettsiae and in the production of rickettsia-free spermatophores. Based on these observations, the mechanism of vertical transmission of the rickettsiae to the progeny occurs only in the female parents. Most rickettsiae in the somatic cells of larvae and adults were coccoid, but some rickettsiae in the ovary and the testis of adult mites showed bacillary forms and were enveloped by a membrane of unknown origin.

Surface morphology of Rickettsia tsutsugamushi-infected mouse fibroblasts.

Sequential morphological changes of the L-cell surface after infection with Rickettsia tsutsugamushi (Gilliam strain) have been examined by scanning electron microscopy and ruthenium red staining technique. Adherence of inoculated rickettsiae to the host-cell surface and their engulfment by the cell were seen at 30 min and have still proceeded at 24 hr post infection (p.i.). Progeny rickettsiae which were lifting up the host cell membrane by budding were observed on the cell surface at 48 hr p.i. The budding of rickettsiae increased gradually in time and, at 96 hr after infection, covered almost the all host-cell surface except of the cell margin. Numerous microvilli observed on the surface of uninfected L-cells decreased gradually p.i.; they had almost disappeared when progeny rickettsiae occurred. Ruthenium red staining specimens clearly showed that the budding rickettsiae were surrounded with the host cell membrane. The following layers were distinguished from outside on: (1) ruthenium red positive fuzzy coat (25 nm thick); (2) a triple-layered cell membrane (5-6 nm); (3) outer and inner leaflets of the rickettsial cell-wall (7-8 nm and 2-2.5 nm, respectively); (4) periplasmic space (15-20 nm); (5) a triple-layered rickettsial cytoplasmic membrane (5-6 nm).

Comparative ultrastructural study on the cell envelopes of Rickettsia prowazekii, Rickettsia rickettsii, and Rickettsia tsutsugamushi.

Rickettsia tsutsugamushi differs from other rickettsiae in its cell envelope organization. The differences were made evident through a comparative study of the outer envelope of R. tsutsugamushi, R. prowazekii, and R. rickettsii by electron microscopy.

Invasion and intracellular growth of Rickettsia tsutsugamushi.

Intracellular multiplication of Rickettsia tsutsugamushi, a causative agent of scrub typhus, was examined by electron microscopy of specimens prepared at various time intervals after infection of in vitro cultured cells. The sequential morphological growth cycle of the microorganism is presented diagrammatically.

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.

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.

Some contributions of electron microscopy to the study of the rickettsiae.

Electron microscopy has provided valuable insights into the study of rickettsiae as intracellular parasites from several important perspectives. This tool has allowed researchers to delineate the fine structural features of these organisms and to show that they truly resemble free-living bacteria. Furthermore, it has been shown that there are subtle, but distinct differences in the outer envelope structure of some members of the genus Rickettsia that may explain reported differences in tinctorial properties and in their sensitivity to certain antibiotics. With Coxiella burnetii, electron microscopy has helped significantly in the characterization of the pleomorphic nature of the organism including formation of terminal bodies that resemble endospores of gram-positive bacteria. Electron microsxopy has also helped to define the relationship of the rickettsiae to their host cells. For example, ultrastructural analysis can reveal whether organisms exist free within the cytoplasm or nucleus (members of the genus Rickettsia), or whether they are bound by a phagosomal or phagolysosomal membrane (Ehrlichia and Coxiella). Finally, although all rickettsiae eventually destroy their host cell, it has been shown through transmission electron microscopy that this destruction might be mediated by different mechanisms that are specific for different rickettsial species.

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.

Assembly of Rickettsia tsutsugamushi progeny in irradiated L cells.

In the assembly of Rickettsia tsutsugamushi progeny in irradiated L cells, nascent forms first appear as undemarcated foci in the host cell granular cytoplasm, in which electron-lucent filamentous (f) and electron-dense granular (g) areas differentiate. Morphological observations indicated that the assembly involves formation of a filamentous network in the f area, manufacture of rickettsial ribosomes in the g area, and formation of mildly electron-dense fuzzy zones, along which a double membrane assembles.

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 heavy 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.

Electron microscopic observations of the embryo Leptotrombidium (Leptotrombidium) pallidum naturally infected with Rickettsia tsutsugamushi.

Embryos of Leptotrombidium (Leptotrombidium) pallidum mites naturally infected with Rickettsia tsutsugamushi were examined by electron microscopy. Rickettsiae were not found in eggs just after oviposition, but were easily detected in cells at the various parts of the embryos just before hatching, indicating that the rickettsiae are surely vertically transmitted from infected adult mites to the larvae through embryos, and the rickettsiae may multiply in situ during the developing process of the embryo.

Growth pattern of Rickettsia tsutsugamushi in irradiated L cells.

Irradiated L cells infected with Rickettsia tsutsugamushi were studied under the electron microscope to define the morphological growth pattern of the organism. For 2 days after inoculation, no rickettsiae were found either extra- or intracellularly; after 2 days multiple rickettsiae appeared within the host cells without morphological evidence of their entry. These observations showed that the rickettsiae within the cell were assembled in situ by segregation of portions of the granular cytoplasm and subsequent internal differentiation and surface membrane assembly of the segregated bodies. The protoplasmic (P) bodies, which seemed to be formed by shedding infected-cell granular cytoplasm, consistently appeared on the surface and within the phagosomes of the host cells. Rickettsiae were occasionally seen entering host cells in the later phase of infection; these were apparently the ones assembled within the P bodies. This suggested that the P bodies, and not the rickettsiae, were the major infectious particles that transmitted the rickettsial genetic substance among the host cells. On the basis of the present morphological observations, viral-type multiplication for R. tsutsugamushi is proposed.

Electron microscopic observations of Orientia tsutsugamushi in salivary gland cells of naturally Infected Leptotrombidium pallidum larvae during feeding.

We performed a detailed electron microscopic observation on the escaping process of Orientia tsutsugamushi from the salivary gland cells of naturally infected trombiculid larvae into the acinar lumen of the gland during feeding on mice. In unfed larvae, many O. tsutsugamushi were intermingled with secretory granules in the cytoplasm of the salivary gland cell. O. tsutsugamushi was neither found in the acinar lumen nor observed escaping from the apical surface of the gland cell. In contrast, in the larvae fed on mice, many O. tsutsugamushi were observable in the acinar lumen. They were enveloped with the host glandular cell membrane. In salivary gland cells, secretory granules changed the distribution and accumulated in the apical region. In such cells, the majority of O. tsutsugamushi were found at the base of the cell. Some O. tsutsugamushi were pushing the glandular cell membrane outward in various degrees, showing different stages of escape. These findings suggest that larval feeding induced O. tsutsugamushi escape from salivary gland cells, that the escape was by budding, during which O. tsutsugamushi were enveloped in the host cell membrane, and that O. tsutsugamushi would be injected into the mouse skin as a mixture with mite saliva. The study also revealed the presence of many small vesicles that had the same cell wall structure as O. tsutsugamushi in the cytoplasm of the salivary gland cell. Most of them seemed to be products from degenerated Orientia.

Identification of the target cells of Orientia tsutsugamushi in human cases of scrub typhus.

Orientia tsutsugamushi is the etiologic agent of scrub typhus, a chigger-borne zoonosis that is a highly prevalent, life-threatening illness of greatest public health importance in tropical Asia and the islands of the western Pacific Ocean. The target cell of this bacterium is poorly defined in humans. In this study, O. tsutsugamushi were identified by immunohistochemistry using a rabbit polyclonal antibody raised against O. tsutsugamushi Karp strain in paraffin-embedded archived autopsy tissues of three patients with clinical suspicion of scrub typhus who died during World War II and the Vietnam War. Rickettsiae were located in endothelial cells in all of the organs evaluated, namely heart, lung, brain, kidney, pancreas, and skin, and within cardiac muscle cells and in macrophages located in liver and spleen. Electron microscopy confirmed the location of rickettsiae in endothelium and cardiac myocytes.