[Electron microscopic determination of the localization of the group-specific antigen of Halprowise (Chlamydiae) by the direct immunoperoxidase method]. / Elektronno-mikroskopichekoe opredelenie lokalizatisii gruppospetsificheskogo antigena gal'provii (khlamidii) priamym immunoperoksidaznym metodom

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.

[Improvement in the method of cultivating Halprowiae (Chlamydiae) in cell cultures]. / Usovershenstvovanie metoda kul'tivnirovaniia gal'provii (khlamidii) v kul'ture kletok.

Comparative evaluation of several methods increasing the effectiveness of cultivating Halprovia (7 strains of different origins) in L-929 and McCoy cell cultures has shown that the maximum effectiveness can be achieved by supplementing the standard method for the cultivation of obligate intracellular parasites with the method of forced adsorption of Halprovia on the monolayer and the treatment of cells with DEAE dextran or cycloheximide. The peculiarities of detecting cytoplasmic halprovian inclusions in infected cells by staining the specimens with fluorescent antibodies, with Lugol's solution, and according to May-Grünwald-Giemsa are described.

[Yield of Chlamydia from an infected cell studied by scanning electron microscopy]. / Izuchenie vykhoda khlamidii iz infitsirovannoi kletki metodom skaniruiushchei élektronnoi mikroskopii.

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.

[Indication of halprowia (chlamydia) antigens by the direct immunoperoxidase method]. / Indikatsiia antigenov gal'provii (khlamidii) priamym immunoperoksidaznym metodom

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.

[Ultrastructure of the initial stages of interaction between halprowiae (Chlamydiae) and host cells]. / Ul'trastruktura nachal'nykh étapov vzaimodeistviia gal'provii (chlamidii) s kletkoi-khoziainom.

The initial stages of the interaction of halprowiae (strain CP-1), isolated in Reiter's syndrome, with a monolayer culture of cells L-929 were studied. The electron-microscopic examination of the cells every 2 hours during the first 24 hours after the inoculation allowed to follow the processes of adsorption, endocytosis of elementary bodies and their transformation into reticulate bodies by the "decondensation" of nucleoid, which indicated the beginning of the vegetative (reproductive) stage of the developmental cycle. During all the periods of observation halprowiae were enclosed into the membranes of phagocytic vacuoles formed by host cells. Besides, in other vacuoles (probably, phagolysosomes) halprowiae at various stages of destruction could be observed. 8--10 hours after inoculation the phagosomes containing reticulate bodies began to merge, thus forming an inoculation the phagosomes containing reticulate bodies began to merge, thus forming an intracytoplasmic inclusion. In this inclusion a new generation of the agent was formed by binary-like fission. At the beginning of the vegetative stage halprowiae seem to be highly sensitive to the action of exo- and endogenic factors which may cause their L-transformation. Depending on the reaction of the host cell at the initial stages of interaction with halprowiae, 3 ways of such interaction are seemingly possible: developmental cycle, destruction in phagolysosomes. L-transformation.

[Study of lysosomal enzymes in chick embryo fibroblasts infected with microorganisms of family Halprowiaceae (Chlamydiaceae)]. / Izuchenie lizosomal'nykh fermentov fibroblastov kurinykh embrionov pri infitsirovanii kletok mikroorganizmami semeistva Halprowiaceae (Chlamydiaceae)

It is shown that infection of chick embryo fibroblasts with agents of paratrachoma and meningopneumonia Halprowiaceae (Chlamydiaceae) causes a sharp decrease of the activities of lysosomal enzymes, e.g. acidic alpha-glucosidase, beta-glucuronidase, beta-galactosidase, alpha-mannosidase, acid phosphatase, etc. The activity of cytosol enzymes (neutral alpha-glucosidase, amylo-1,6-glucosidase) does not change, however. A decrease in the activities of lysosomal enzymes in infected fibroblasts occurs some time later after inoculation and is due to a release of lysosomal enzymes from the fibroblasts into the culture medium, without loss of cell integrity. No changes in the activity of lysosomal enzymes in fibroblasts and culture medium is observed in the case of inoculation of cells with a killed agents, as well as after contact of cells with a suspension of normal chick embryo yolk sacs. The release of lysosomal enzymes from halprowiae-infected chick embryo fibroblasts probably occurs by the exocytosis.

[Action of penicillin on the causative agent of paratrachoma in the dynamics of an in vitro infection]. / Deistvie penitsillina na vozbuditel' paratrakhomy v dinamike infektsii in vitro.

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.