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The study of the monolayer cell culture L-929, infected with strain CP-1 isolated from a patient with Reiter's syndrome, by means of scanning electron microscopy demonstrated that the release of chlamydiae from infected cells occurred in two ways: (1) by active ejection, probably as the consequence of the local rupture of the host cell, and (2) by more quiet "eruption" due to rupture in the plasmolemma . Elementary chlamydial bodies released from the cell had a spherical form, and reticular bodies had an irregularly spherical form, often with recesses and digitiform protrusions.

The direct immunoperoxidase technique was applied to the study of the localization of a group-specific antigen of Halprowiae (Chlamydiae) in the causative agents of meningopneumonia (strain MP) and of paratrachoma (strain LB-I) at the individual stages of their developmental cycle in the L-cell (16, 24 and 48 hours after infection of the culture). The product of reaction to peroxidase pointing to the localization of the group-specific antigen was localized not only on the surface, but in the whole thickness of the cell wall of the initial bodies in the form of an even electron dense layer, 200--280 A in thickness. A weak positive reaction was also noted on the outer layer of the sytoplasmic membrane. The periplasmic space remained free of the reaction product. Localization of the reaction product in the intermediate and elementary bodies remained unchanged. At the initial stages of the developmental cycle (16 hours after the infection) the reaction) the reaction product was revealed not in the whole cell wall, butin some of its areas only. There were found no qualitative differences in the localization of the group-specific antigen in the Malprowiae strains under study.

Monolayer cultures of L-cells (mouse fibroblasts) were inoculated with the causative agent of paratrachoma (strain LB-I). Simultaneously penicillin in doses of 0.01, 0.1, 1, 10, 25, 50, 100, 150 and 200 microgram/ml was added and its effect on the causative agent in the infection dynamics (18, 24, 48 and 72 hours after the inoculation) was studied with the light and electron microscopes. Gradual changes in the ultrastructure of the vegetative forms were observed by the 24th hour with the use of penicillin in a dose of 0.01 microgram/ml: the size of the vegetative forms increased, the cell wall membranes separated and periplasmic space significantly enlarged, the protoplast fragments disjoined into it, large forms vacuolized and were fragment with membranes, sometimes multilayer ones. When the culture was exposed to large doses of penicillin, the rate of the changes in the structure was higher and they were simultaneously of several types. Various types of the changes and possible modes of their formation were analyzed. Morphologically they are similar to the processes observed in L-transformation of bacteria. However, these structures were not infectious.

The surface structure of chlamydial elementary bodies (strain PL-B577 causing enzootic abortion of ewes and strain CP-1 isolated from a patient with Reiter's syndrome), concentrated directly on the grid of an electron microscope, was studied by the method of negative staining. The surface of the cell wall of chlamydial elementary bodies in the preparations of crude cultures, not subjected to enzymatic purification, was formed by spherical subunits 4 nm in diameter. The surface of the cell wall of elementary bodies was found to have projections 10-20 nm long and 3-4 nm in diameter, as well as annular structures 15-22 nm in diameter in hexagonal arrangement, spaced at 45 nm and probably traversed by these projections. The number of such structures in a group was 12-15. These data correspond to the model representing the structure of the surface of chlamydiae, which was proposed by Matsumoto in 1979.

The authors carried out a comparative electron-microscopic study of the ultrastructure and the developmental cycle of 15 Halprowiae (chlamydiae) strains isolated in different pathology from man and animals. Halprowiae were cultivated in the yolk sac epithelium of the developing chick embryos and in the L-cell monolayer cultures without any special action of the antibiotics and medicines. Formation of minute round structures ("minute forms"), 50--120 nm in diameter, separating from the protoplast into the periplasmic space and surrounded with cytoplasmic membrane by the vegetative forms of a number of strains (MP, AP-23, 25-SM, LB-1, MyB), is described. Along with minute forms, Halprowiae formed polymorphous giant bodies with long protrusions into the inclusion cavity; there was also a separation of vesicles of the cell wall membrane. This apparently indicated a structural derangement of the cell wall in these vegetative forms of Halprowiae. On the basis of similarity of the mentioned structures detected in Halprowiae reproduction to the known morphological characteristics of the L-form bacteria a possibility of L-transformation in Halprowiae during the normal developmental cycle as a peculiar compensatory adaptation to the action of host cell is supposed.

The authors modified and approbated the direct immunoperoxidase method for the indication in the infected L cells of the halprowia (chlamydia) antigens--etiological agents of trachoma, ocular and urogenital forms of paratrachoma, arthritis, meningopneumonia and enzootic abortion of sheep. Different types of localization of the group-specific halprowia antigen were revealed in the cytoplasm of the affected cells by light microscopy; electron microscopy demonstrated localization of this antigen in the membrane of the cell wall of the elementary and initial bodies. Specificity of the test-system used, no lesser sensitivity than the sensitivity of direct immunofluorescent method of indication of the same microbies, with a possibility of detection of stably stained antigens by widely accessible light microscopy underly practical usefulness of direct immunoperoxidase method of the diagnosis of halprowiosis (chlamydiosis) of man and animals.

Morphology and ultrastructure of Halprowia arthritidis, strain SR-1 (HSR), isolated from the synovial fluid of a patient with Reiter's syndrome, was studied in the membranes of the yolk sacs of the developing chick embryos and the L-cell culture. In acridine orange staining for light and fluorescent microscopy there was revealed intracellular cytoplasmic inclusions containing HSR structures at various stages of its reproduction characteristic of halprowia (chlamydia). The direct immunofluorescent method demonstrated the presence of a characteristic HSR antigen not only in the developed inclusions, but also at the early stages of infection, when the morphological HSR structures could not be found by light microscopy. The ultrastructure of the HSR inclusions and forms in the cycle of development (of the initial and elementary bodies) of the SR-1 strain was typical of other halprowia. A peculiar structure of a complex of cell wall and cytoplasmic membrane of the elementary body was described. Taking into consideration the biological characteristics of HSR revealed earlier it can be considered to be a typical representative of Halprowiales s. Chlamydiales. The data obtained on other halprowia, pointing out the fact that criteria of compactness and diffuseness of inclusions, the presence of absence of glycogen in the inclusions could not serve as taxonomic signs in classification of halrpowia, were confirmed on a model of the SR-1 studied.

The method of cryoultramicrotomy was adapted for the study of the ultrastructure of HeLa and McCoy cells in monolayer cultures infected with Chlamydia, obligatory intracellular procaryotic parasites, the causative agents of ornithosis (strain Loth) and paratrachoma (strain LB 1). The cryosections were obtained by the fixation of the monolayer with 2.5% glutaraldehyde, by the gradual infiltration of precipitated cells with sucrose (0.6--1.2--1.8--2.3 M) prior to freezing in liquid nitrogen, and by the treatment of sections with 1% aqueous methyl cellulose solution before drying. This method ensured good preservation of both Chlamydia, in intracytoplasmic inclusions and host cells, as well as regular reproducibility of the results. Ultrathin sections showed a considerable polymorphism in the vegetative forms of Chlamydia, which was probably due to the structure of their cell walls. Chlamydia, were found to form small vesicle-like structures in the cavities of inclusions. The cell walls and granules inside the elementary bodies of the causative agent of ornithosis were stained with the use of phosphotungstic acid--HCl, pH 0.5.