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.     

Electron microscopic observations on tracheal epithelia of mice infected with Bordetella bronchiseptica

To clarify the pathogenesis of Bordetella in vivo infection, the tracheal epithelia of mice were examined in detail by electron microscopy at various intervals after intranasal inoculation with graded doses of phase I Bordetella bronchiseptica. In mice infected with a lethal dose (6 to 7 x 10(7) CFU), a remarkable rupture of the cell membranes of cilia and microvilli of the middle trachea was found on day I postinfection. The rupture of the membrane was observed over the entire tracheal epithelia, on day 2 after infection. The affected cilia were constricted at the transitional region and were broken off. In the ciliated cells the adherence of organisms to ciliary apexes and colonization in the interciliary spaces were also remarkable. In both the ciliated and nonciliated epithelial cells, the cytoplasmic vacuolation and pyknosis or karyorrehexis were also notable. In mice infected with one-tenth of the lethal dose, similar findings were seen, but appeared more slowly and the bacteria were not seen attaching to ciliary apexes. In mice receiving one-hundredth of the lethal dose, only mild cilial abnormality such as aggregation of cilia, and slight cytoplasmic vacuolation were found 6 days postinfection. Based on these findings, a possible mechanism of the ciliary damages produced by B. bronchiseptica was postulated.     

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 on ciliated epithelium of tracheal organ cultures infected with Bordetella bronchiseptica

Using mouse tracheal organ cultures, the pathogenic effect of Bordetella bronchiseptica to epithelial cells was studied by electron microscopy. The ultrastructure of epithelial cells in uninfected tracheal rings was preserved well for longer than 3 days. In mouse tracheal rings infected with graded doses (3 x 10(5) to 10(7) CFU/ml) of phase I B. bronchiseptica, the colonization in the interciliary spaces of ciliated epithelial cells was observed after a 20-hr infection period. The infected tracheal rings showed swelling of nonciliated cells as well as ciliated cells, rupture of cell membrane of cilia, swelling and disappearance of cilia, and atrophic cytomorphosis of epithelial cells. The severity of these changes occurred depending on the infection doses. These changes were essentially similar to those observed previously in the tracheal epithelia of the B. bronchiseptica-infected mice. The usefulness of this in vitro model was suggested for studying the pathogenesis of Bordetella infection.     

Transovarial Transmission of Orientia tsutsugamushi in Leptotrombidium palpale (Acari: Trombiculidae)

Transovarial transmission of Orientia tsutsugamushi in colonies of Leptotrombidium palpale was studied in the parent and F₁ and F2 generations. Both transovarial transmission and filial infection rates were 100% in the parent and F₁ generations of Leptotrombidium palpale. The filial infection rate in the F₁ generation was 100%, but it declined to 94.3% in the F₂ progeny. The sex ratio of the F₁ generation from infected L. palpale was 1∶0.8 (male:female) and the proportion of males was relatively high. This study is the first to report on the transovarial transmission of O. tsutsugamushi in L. palpale. High transovarial transmission rates in L. palpale suggest that this species might be one of the major vectors of tsutsugamushi disease in Korea.     

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.     

Scanning electron microscopic observations on the 9-day opossum (Didelphis virginiana) embryo

All three germ layers are present in the opossum embryo by the 9th prenatal day. The embryo proper is part of, and continuous with, the remainder of the chorionic wall. The wall of the yolk sac-chorion away from the embryo consists only of an outer covering of ectoderm and an inner layer of endoderm. Ectodermal cells covering the neural folds have dome-shaped apices and often show large, bleb-like expansions. Microvilli are short and few in number. The apical surfaces of ectodermal cells that overlie the parietal mesoderm are relatively smooth and show scattered, short microvilli that tend to be concentrated at cell junctions. The apices of ectodermal cells that cover the extraembryonic region are more rounded, and the cells balloon from the surface. Each cell shows abundant elongate microvilli and occasional cytoplasmic blebs. Endodermal cells that line the chorion and form the third (innermost) layer of the embryo are similar in their surface morphology.     

Electron microscopic observations of FL cells infected with herpes simplex virus. III. Dense bodies.

FL cells infected with the -GCr Miyama strain of herpes simplex virus at an adsorbed multiplicity of approximately 10 were fixed at late stages of infection and examined by electron microscopy. Dense bodies containing electron-dense material and surrounded by a limiting membrane were occasionally observed in the perinuclear disternae and in intranuclear vacuoles. Budding of electron-dense material to the cisternae with acquisition of a limiting membrane at the inner nuclear membrane was also occasionally observed. These findings are in constrast with observations that in cells infected with cytomegalovirus numerous dense bodies and their budding process were observed only in the cytoplasmic area.