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Improving the management of children with asthma associated with mycoplasma infection is important. Aim: To study the duration of the persistence of antigens, and DNA in a free state, in the structures of circulating immune complexes (CICs) and living cells of Mycoplasma pneumoniae (Mpn) and Mycoplasma hominis (Mh) in children with asthma. In total, 205 children with asthma from 1 to 14 years were observed. The reaction of aggregate-hemagglutination (AHAA), the direct immunofluorescence reaction (DIF), the reaction of the polymerase chain reaction (PCR), and the culture method were used. In addition, 47 children were re-examined 1.5 months after the treatment of mycoplasma infection with azithromycin. The number of samples positive for antigens and DNA in the free state and in the structures of CICs significantly decreased. Then, 50 blood serum samples containing Mh antigens, and 50 samples containing Mpn antigens were analyzed by culture method. Mh was isolated in 21 (65.5%) of 32 samples containing DNA. Mpn was isolated from antigen-positive samples in nine cases. The presented data indicate the long-term persistence of antigens, and DNA of mycoplasma cells in the free state, in the structure of CICs, as well as in the form of "microcolonies". A high level of CICs can be used to predict the course of the disease and the response to therapy.

Introduction. The Mollicutes class unites cell wall lacking bacteria many of which are membrane parasites and opportunistic bacteria. Aim . This study describes a novel morphological form found in the five species belonging to the bacterial class Mollicutes, and referred to as microcolonies (MCs). Methodology. MCs were obtained as described below and characterized with bacteriological and immunological methods, and microscopy. Results. In contrast to typical colonies (TCs), MCs are characterized by tiny propeller-shaped colonies formed by rod-like cells tightly packed in parallel rows. These colonies were observed within routinely cultivated cultures of type strains 7-12 days post-plating. Rod-like cells were visualized using a scanning electron microscope within TCs with a 'fried-egg-like' appearance. MCs were not observed to revert to TCs. MCs were resistant to antibiotics and other treatments effective against TCs. Pure MC cultures were generated in vitro by treatment of Mycoplasma cultures with hyperimmune serum, antibiotics or argon non-thermal plasma. MCs of Mycoplasma hominis strain H-34 were characterized in detail to confirm that they belonged to that species. MCs tested positive via PCR with M. hominis -specific primers, direct fluorescence and epifluorescence tests, and Western blotting with the camel-derived nanobody aMh-FcG2a, which is specific to the MH3620 transporter protein. Meanwhile, MCs behaved differently in standard bacteriological tests. Pure MC cultures were also isolated directly from clinical samples of the serum, synovial liquid and urine of patients within flammatory urogenital tract diseases, asthma or arthritis. In total, 79 independent MC cultures were isolated from clinical samples including M. hominis ( n =70), Mycoplasma pneumoniae ( n =2), Mycoplasma fermentans ( n =2) and Mycoplasma spp. ( n =5). Conclusion . MCs play an unknown role in infection pathology and display prominent antibiotic resistance, making them a challenge for the future studies on Mollicutes.

Aim: Study the frequency of detection of mycoplasma and ureaplasma in clinical material from urolithiasis patients., Materials and Methods: Clinical material samples (blood sera, urine, uroliths) from 31 urolithiasis patients were obtained during operations of urolith-removal. Cultural method, LAR and PCR were used in the study., Results: The study of clinical material from 31 patients by PCR has shown, that in 25 individuals. (80.6%) DNA of mycoplasma and ureaplasma was detected, and mycoplasma DNA was more frequently detected in uroliths and less--in-blood sera. Mycoplasma hominis DNA was detected in clinical material of a significantly largerninmber of patients. 23 cultures were isolated from 8 patients by a cultural method, that were identified by PCR as M. hominis. All the isolates have grown as "mini colonies". Even after multiple passages in agar medium, reversion of "mini-colonies" into colonies with a classic morphology was not obtained., Conclusion: A high frequency of detection of mycoplasma and ureaplasma in clinical material of patients with urolithiasis was established. The isolated M. hominis cultures have only grown as "mini-colonies". The phenomenon discovered could give evidence on high variability of mycoplasma and a possibility of existence of previously unknown form of their persistence in human organism.

Aim: Use of a complex of methods for etiologic deciphering of an acute respiratory infection., Materials and Methods: Clinical samples of blood sera, nasopharynx washes and sputum were obtained from 35 patients with acute respiratory disease (ARD). "Difco PPLO Broth" was used for M. pneumoniae cultivation. AHR, IFR, PCR, IFA were used in the study., Results: Results of the study have shown that M. pneumoniae antigens in blood, sera samples were detected in AHR in 32 patients, and specific G and M class antibodies--in 21 and 18 cases, respectively. Simultaneous detection of IgG and IgM was registered in 14 patients. M. pneumoniae cell DNA was detected in 10 of 20 blood sera samples. Circulating immune complexes were isolated from blood sera of 8 patients (4 with pneumonia, 4 with ARD) and M. pneumoniae antigens were detected in them by using direct-IFR. IFR study of sputum and nasopharynx smears has shown that M. pneumoniae antigens were detected in 29 of 35 samples. In 12 of 15 smear samples M. pneumoniae. DNA was detected by PCR. In 10 cases results of antigen detection by IFR as well as DNA in PCR coincided. Results of analysis of all the clinical material have shown that in 33 of 35 patients positive results coincided for 2 or 3 and in some cases 4 of the laboratory study methods used., Conclusion: The use of diagnostic test complex significantly increases the accuracy of the study results, and detection of specific antibodies allows to determine disease period.

Aim: Establishment of ratios that would allow to execute recalculation of mycoplasma concentration from CFU/ml and/or CCU/ml into units obtained during PCR analysis--geq/ml., Materials and Methods: Pure cultures of Mycoplasma hominis, Ureaplasma parvum and Ureaplasma urealyticum were studied by cultural and molecular-biological methods with quantitative evaluation. Studies of initial cultures as well as series of 10-fold dilutions were carried out. 32 experiments in total were carried out., Results: Ratio between geq/ml and CFU/ml for M. hominis was 3.5; geq/ ml and CCU/ml ratio--4.4. Ratio between geq/ml and CCU/ml for U. parvum was 7.1; for U. urealyticum--11.2., Conclusion: Ratios between indexes obtained during quantitative study of pure genital micoplasma cultures by using 2 methods were established.

Aim: Study of possibility of generalization of mycoplasma infection in patients with urogenital pathology., Materials and Methods: Among the examined patients 5 males characterized by risky sexual behavior with pronounced symptoms of infection or without those were selected. Patients were examined by a complex of methods for the presence of mycoplasma infection by culture, PCR, DFA, PHA, AHR and by detection of specific immune complexes in blood sera. Scrapes from urogenital tract, blood sera samples, urine, saliva, prostatic fluid were materials for the study., Results: In blood of all patients in ELISA antibodies against Mycoplasma hominis were detected; in PHA they were detected only in 2 individuals. In all the patients in blood CIC were detected including antigens and DNA of one or several mycoplasma species. Sperm of 3 individuals was infected by Ureaplasma spp., 2--M. genitalium. In saliva of 2 individuals M. hominis was detected, 3--U. urealyticum., Conclusion: In all the examined patients the infection was shown to have generalized character. This phenomenon presents itself as quite significant because mycoplasma may cause anti-apoptotic and oncogenic effect.

Aim: Study the possibility of prolonged conservation in macroorganism of antigens, mycoplasma cell DNA and live pathogen cells as part of CIC against the background of persisting antigen biostructures., Materials and Methods: Aggregate-hemagglutination, direct immunofluorescence reactions and PCR method were used to determine antigens and DNA. Circulating immune complexes from blood sera samples were isolated by M. Digeon et al., mycoplasma isolation from CIC was carried out in SP-4 medium, species identity of the isolated mini-colonies was confirmed by real-time PCR method., Results: In patients with urogenital and respiratory pathology the frequency of detection of Mycoplasma hominis, Ureaplasma urealyticum and Mycoplasma pneumoniae in free state was 63.3, 53.1 and 80.82% of cases, respectively. Specific CIC in patients with verified respiratory mycoplasmosis 1 month after the onset of the disease were registered in patients with severe course of the disease, bronchitis and diseases of upper respiratory tract--in 92.5, 74.7 and 25.7% of cases, respectively. In children, bronchial asthma patients the frequency of detection of antigens and DNA of M. pneumoniae cells in free state was 72.6 and 12.33%, as part of CIC--in 60.27 and 43.8% of cases, respectively. Antigens and DNA of M. hominis in blood of this group of patients were detected in 32.9 and 26.02%, as part of CIC--in 53.42 and 52.05% of cases, respectively. During repeated examination of 12 children after etiotropic therapy execution (generally in 1.5 - 6 months) in 75% of cases antigens of both M. pneumoniae and M. hominis were detected in free state and as part of CIC. DNA of cells of these mycoplasma species were detected in 20 and 33%, as part of CIC--in41.6 and 50% of cases, respectively. In 5 patients after 6 months (after 1 year in 1 case) mycoplasma antigens and DNA were identified in CIC or in blood sera. During cultivation of CIC components precipitated from 5 blood samples of patients of this group containing M. hominis DNA, culture of M. hominis mini-colonies were isolated in 4 cases., Conclusion: The possibility of prolonged persistence of antigens, DNA and whole mycoplasma cells in both free state and as part of CIC in patients with respiratory and urogenital pathology was shown. CIC are thus a peculiar depot, a place of conservation of not only various mycoplasma cell components, but also live cells.

Aim: Study previously unknown forms of persistence of Mycoplasma hominis in host organism., Materials and Methods: Culture method was used for detection of mycoplasmas. Identification was carried out by serological, electron microscopy methods, classic PCR and real time PCR; circulating immune complexes (CIC) were isolated by PEG precipitation., Results: Classic micoplasma cultures could not be isolated from blood even once. At the same time "mini-colony" cultures composed of mini-cells that were hardly passaged but sometimes formed continuous layer of the same colonies were isolated from blood serum samples with high frequency. During reseeding for more than 1 year they never acquired classic form. Not only antigens of M. hominis but its DNA were shown to be present in CIC. Viable cells forming "mini-colonies" identical to those isolated from blood sera were isolated from circulating immune complexes. A system of evidence on identity of isolated M. hominis cultures is presented. Cultures had infectivity and an ability to persist in organs of experimentally infected mice., Conclusion: The isolated forms are apparently the result of adaptation of mycoplasmas to humoral immunity factors.

The antigens, DNA and RNA of mycoplasmas are preset in the blood serum of persons infected with urogenital mycoplasmas. The planting of patients' tests of blood serum containing antigen M. hominis on the artificial growth mediums resulted in the growth of mini-colonies of mini-cells (20-50 nm). The colonies subcultured hardly but sometimes formed solid bacterial lawn though never acquired "fried-egg" classical mycoplasma form. The proofs of identity of these colonies to M. hominis are presented. The mini-cells possessed infectiousness and ability to persist on a long-run in the internal organs of experimentally infected mice. Apparently, mini-cells are formed under impact of stress factors of the host immune defense and they are one of forms of mycoplasma's persistence in human organism.

Aim: To study the time of preservation of antigens and DNA of urogenital mycoplasmas in circulating immune complexes (CIC) in blood of rabbits after single inoculation., Materials and Methods: Rabbits were inoculated with Mycoplasma hominis and Ureaplasma urealiticum cell cultures washed in fetal calf serum. Reaction of aggregate-hemagglutination, immunofluorescence assay and PCR were used for detection of mycoplasmas' antigens and DNA., Results: It was shown that DNA and antigens of M. hominis persist in free state and in structure of CIC during 1 month and 3 months respectively. In immunized rabbits antigens and DNA of mycoplasmas were detected in CIC structure even 6 months after the last immunization. Pattern of detection of DNA and antigens of U. urealyticum in blood of inoculated rabbits consists in that both DNA and antigens of the microorganism were detected in structure of CIC in blood samples during 70 days, whereas in free state they were detected only during 35 days. Incomplete elimination of CIC is possibly related to their small size (11S and lower) that allows them to circulate for a long time., Conclusion: Prolonged persistence of antigens and DNA of mycoplasmas in CIC structure is a fact that requires refinement of diagnostic criteria used for control of effectiveness of etiotropic therapy.

Aim: To study the possibility of existence of antigenemia during urogenital mycoplasmal infections by detection the antigens of agents in blood and viscera of infected animals., Materials and Methods: Rabbits and mice were intraperitoneally inoculated with Mycoplasma hominis and Ureaplasma urealyticum, their antigens and DNAs. Samples of blood and visceral organs were studied by several methods: cultural with use of standard media, PCR, RT-PCR, indirect hemagglutination test, and immunofluorescence assay for detection of antibodies., Results: Bacteremia with M. hominis develops during 2 months after inoculation in rabbits and 3 weeks after inoculation in mice. Antigens of M. hominis and U. urealyticum were detected in serum and visceral organs significantly frequently than live cells and DNAs. Prolonged preservation of the antigens in animals' blood and viscera after intraperitoneal administration of "pure" antigens points to the presence of true mycoplasmal antigenemia. Forms of existence of antigens in organism are different-they can represent corpuscular antigens as well as soluble molecular compounds circulating in blood both in free state and in structure of immune complexes. Antigens as well as live cells are preserved in all studied organs., Conclusion: Inoculation of rabbits and mice with M. hominis or U. urealyticum resulted in development of generalized infection with persistence of the agent in all studied organs during initial phase of infection and predominant persistence in organs of immunogenesis during later phases.

How long the viable cells of M. hominis, Ureaplasma spp., U. urealyticum, U. parvum, and their antigens retained in human serum at 37 degrees C was investigated. M. hominis cells were shown to hold their viability within 12 days with a gradual titer drop, the antigens being also detected within 12 days whereas intracellular and extracellular DNAs were seen within 40 days (an observation time). Under the same conditions, Ureaplasma cells died after 24 hours, their antigens were disrupted following 3 days and intracellular and extracellular DNAs of different species were detectable by polymerase chain reaction (PCR) within 17-40 days. The long preservation of extracellular and dead cell DNAs suggests that diagnostic examination of patients by means of PCR may yield false-positive results.

The authors compared the detection rate of DNA of urogenital Mycoplasmas (Ureaplasma spp. and M. hominis) in the urogenital tract (UGT) and serum samples by polymerase chain reaction. Testing the smears and serum samples from the same patients (n = 112) showed that Ureaplasma was more frequently detected in the UGT smears than in the serum samples. There was the same trend towards M. hominis although the difference was not so significant. Therefore, the detection of these microorganisms in UGT is of more informative value for diagnostic purposes. Examination of the serum samples for these purposes provides useful information in understanding the mechanisms of generalization of Mycoplasma infections and studying the routes and modes of spreading these bacteria in the organism.

Six different methods have been employed to detect M. hominis (Mh) and U. urealyticum (Uu) in clinical samples collected from 67 men. The results obtained by PCR and IF test were approximately equal: 13.6 and 13.44%--Mh and 44.4 and 48.8%--Uu, respectively. Mycoplasmas were detected by cultural method less frequently (9.6%--Mh, 32.2%--Uu). The highest infection rates were obtained in the test for blood antigens (40%--Mh and 63%--Uu). At present a commercial diagnosticum to detect mycoplasma antigents in blood is lacking. Sometimes the results of cultural method are positive, while the PCR results are negative. So the optimal scheme based on both PCR and culture has been proposed.

The effect of different physical and chemical factors on the process of transition of persisting mycoplasmas into active virulent state was studied to find out conditions promoting the development of acute infection in the course of chronic infection. The activity of the gene coding the synthesis of protein P1, the main pathogenicity factor, was evaluated in the reverse transcriptase test--PCR under conditions of heat and cold shocks, oxidation stress, varying osmotic pressure. An increased osmotic pressure, heat and cold shocks were shown to induce the transcription activation of the gene coding the synthesis of P1 in the avirulent strain, and consequent restoration of its adhesive properties indicative of virulence. M. pneumoniae avirulent strain was characterized by greater resistance to oxidation stress and to a rise in osmotic pressure, this property requiring further study. Quite probably, M. pneumoniae DNA-binding proteins earlier detected in persisting and avirulent cultures by taking part in the compactization of DNA contribute to the adaptation of mycoplasmas to different stress influences. There are grounds to suggest that M. pneumoniae possess mechanisms regulating the expression of genes, in particular the gene coding the main pathogenicity factor, under the influence of different environmental factors.

Three isogenous strains M. pneumoniae, i.e. virulent FH, avirulent FH400 and a revertant with a restored virulence (FHR) and isolated from an avirulent strain, were studied. The mechanism of hemadsorption and the ability to cause an infection in Syrian hamsters were found to be damaged in the avirulent strain. The detection of a specific mRNA by the RT-PCR method showed, apart from the loss of the main adhesin (protein P1), a lack of general components of the phosphoenol-pyruvat-dependable phosphotranspherase system (PTS), i.e. enzyme 1 and protein HPr. The recovery of virulence by passing an attenuated strain through animals with induced immunodeficiency correlated with the recovery of the activity of a gene encoding the P1 adhesion protein and with the onset of the PTS function activity. An analysis of published data was made use of to try to detect a correlation between the functional PTS activity in cell and virulence of M. pneumoniae.

To create the controlled model of respiratory mecoplasmosis, laboratory animals with induced immunodefeciency were infected with M. pneumoniae strain having different degrees of virulence. Immunodeficient state was induced in susceptible animals (hamsters) by the injection of cyclophosphamide. The infection of immunodeficient animals with a virulent strain induced the development of severe manifest pneumonia with 50% mortality rate. The infection of the animals with induced immunodeficiency with the avirulent strain attenuated in the process of 10-year subculturing in acellular media led to the development of moderate pneumonia characterized by the proliferation of the infective agent in pulmonary tissues and the presence of pathomorphological changes. The simultaneous infection of control hamsters with the same strain did not induce the development of infection. The infection of the experimental animals with the avirulent strain in the presence of induced immunodeficiency resulted in the partial restoration of the virulent properties of the strain, which was manifested by the activation of the capacity of the infective agent for colonization and proliferation in body tissues of the animals.

The complex of investigations was carried out with a view to study the mechanisms making it possible for mycoplasmas to switch off the expression of definite genes in persistence. The main method used in these investigations was the method of molecular portrayal, or RNA fingerprinting, with the use of arbitrary primers. The comparative analysis of gene expression in M. pneumoniae strains having different virulence revealed that in the avirulent strain subcultured under in vitro conditions the expression of considerable number of genes was inhibited. In the body of an experimental animal the expression of many genes, including the gene coding the synthesis of the main pathogenicity factor, adhesion protein, switched on.

The preparative scheme for the purification of proteins with molecular weights of 39 and 79 kD, obtained from L. monocytogenes membrane fractions, has been developed. This technology included the cultivation of bacteria in heart-brain broth, isolation of bacterial membranes, the extraction of their components with Triton-X-100 and chromatography on Superose columns. The purified proteins have been shown to form structures with a molecular weight of 500-100 kD and pl 4.7 in water solutions.

A panel of mAb (IgG1, IgG3, IgM) against Legionella pneumophila cytolysin (CL)-protease of 37 kDa was obtained. Subtyping of L. pneumophila strains of serogroup 1 by using mAb against CL (mAb-CL) was carried out. The results of comparative analysis of the specificity of mAb-CL and the panel of mAb kindly provided by Dr. J. M. Barbaree (Centers for Disease Control, Atlanta, GA) allowed us to recommend mAb-CL to be used as a diagnostic tool to reveal the pathogenicity of L. pneumophila strains of serogroup 1. Hybridomas were also raised in a syngenic system which produced anti-idiotypic mAb (mAb2) against anti-CL mAb B6/1. The Ab2 belonged to Ab2 gamma type: 1) Ab2 reacted with B6/1 Id only, 2) Ab2 inhibited the interaction of B6/1 Ab1 with CL, and 3) CL inhibited the reaction of Ab2 with Ab1. The use of Ab2 allowed us to show that B6/1 Id is expressed in 4 to 32% of serum antibodies during the primary and secondary immune responses of BALB/c mice to CL. Ab2 induced the production of anti-anti-idiotypic antibodies (Ab3) in BALB/c mice, and some of them reacted with CL. Thus, we have demonstrated the possibility of inducing an antibody response to CL (one of the main L. pneumophila pathogenic factors) in intact syngenic mice with anti-idiotypic antibodies.

Studies on the typing of L. pneumophila strains of serogroup 1, isolated from patients and environmental objects, have been made with the use of monoclonal antibodies (McAb) to cytolysin. The results of the comparison of the specificity of our McAb with that of a commercial set McAb obtained from the USA make it possible to recommend preparations based on McAb to cytolysin for the detection of L. pneumophila pathogenic strains of serovar 1. The use of FITC- and peroxidase-labeled McAb to cytolysin permits the reduction of the time necessary for the diagnosis of Legionella infections and the detection of the antigen in a dose of 10 ng/ml.

The study of the biological properties of monoclonal antibodies (McAb) to L. pneumophila cytolysin has been carried out. These McAb have been shown to possess no capacity for the in vitro neutralization of cytolysin and the protection of guinea pigs from aerosol infection with L. pneumophila. Still the protective effect of the McAb under study has been observed in experiments with the intraperitoneal infection of guinea pigs, which is indicative of the possibility, in principle, of involving humoral immunity into the protection of the body from Legionella infection.

The immune structure of different professional groups with respect to L. pneumophila has been studied. A wide spread of Legionella infection among subway construction workers, subway personnel and railroad conductors has been established, which makes it possible to regard these professions as high risk groups susceptible to Legionella infection. The risk factors for this group are as follows: work in the atmosphere of high dust content with air supply by means of air conditioners, contact with soil in the process of earthwork. The bacteriological study of water samples obtained in dining cars has resulted in the isolation of two strains of L. pneumophila, serogroup I, which confirms the possibility of cases of Legionella infection among railroad personnel.

L. pneumophila virulent culture and the filtrate of this culture disintegrated with ultrasound were shown to be toxic for guinea-pig peritoneal macrophages in vitro and for AKR mice. The virulent culture of this infective agent and the filtrate of the supernatant fluid obtained from the washings of its virulent culture had no toxic effect on a macrophage monolayer culture and on mice. The use of these models for characterizing Legionella intracellular toxic activity, as well as for characterizing Legionella strains isolated from patients and the environment, is proposed.

The fusion of spleen cells, taken from BALB/c mice immunized with the purified preparation of L. pneumophila cytolysin, with cells Sp2/0 and NP has been carried out. As a result, hybridoma cells producing IgG1, IgG3 and IgM antibodies to this protein have been obtained. All monoclonal antibodies (McAb) thus obtained react with L. pneumophila strain lysates in the precipitation test, while IgG3 and IgM antibodies react with erythrocyte diagnostic agents prepared from the lysate of L. pneumophila cells in the hemagglutination test. In the Western blot assay, McAb react with the 37 KD protein (cytolysin) and a number of other proteins from L. pneumophila cultures and L. pneumophila cell lysate, but do not react with the species-specific protein with a molecular weight of 29 KD, contained in the outer membrane of L. pneumophila, as well as with other species: L. bozemanii, L. dumoffii, L. longbeachae, L. micdadei. The possibility of using these McAb conjugated with FITC and peroxidase for the rapid diagnosis of Legionella infection is shown.

An indirect modification of the radioimmunoassay (RIA) was shown to be highly sensitive in the determination of the minimal amounts of R. prowazekii and their antigens in the organs and tissues of infected animals. RIA proved to be effective in the determination of the antigens in the blood in cases of R. prowazekii persistence in infected animals. The data obtained in these investigations point to good prospects for using RIA in the study of persistence of microorganisms and their antigens.

The electron microscopic study of the interaction of R. akari, strain CK, with the monolayer culture of L-cells was made 4 days after inoculation. Rickettsiae multiplied by transverse binary fission immediately in the cytoplasm of the cells and left the cells by gemmation, surrounded with plasmolemma and a fragment of the host cytoplasm. Alongside with multiplying rickettsiae, spheroplast-like rickettsiae and rickettsiae at the stage of destruction were regularly observed in phagolysosomes. The authors suggest that the normal interaction of rickettsiae with the host cell may be realized by three following routes (1) reproduction, (2) destruction in phagolysosomes and (3) formation of altered (anomalous) forms. The ability of the vegetative forms of rickettsiae and chlamydiae to yield spheroplast-like forms (the initial phase of bacterial L-transformation) indicates that these organisms are similar to bacteria and cannot be themselves regarded as L-forms.

The article presents the results of a study of Legionella pneumophila morbidity in Lithuanian meat-processing factory workers, as well as the specific position of legionellosis in the fever diseases structure among the workers of the industry. The technique showed that Legionella pneumophila 1st serogroup seropositive reaction was traced among the healthy workers in 0.2% (antibody titer 1:64). Rather high percentage (7.1%) of persons with specific antibodies was found among those who had suffered the fever disease 6 month before the examination. Among 191 fever patients examined, in 35 (18.3%) cases antibodies against L. pneumophila were detected in diagnostic titers. In 26 workers legionellosis was diagnosed as a result of serologic testing, and Pontiac fever cases were predominant. 1 case was subsequently followed by pneumonia. A chronic nature of epidemic manifestations of legionellosis was established. The pathogenic antigen was detected in 4 of 32 water samples taken from the factory water pipes.

Alike to macrophages from intact animals, reproduction, destruction and formation of spheroplast-like forms were observed in macrophages from immune guinea pigs 2 months post-infection (p.i.) with the virulent Breinl strain of Rickettsia prowazekii. Unlike to the former, immune macrophages revealed phagolysosomes which were larger in size and contained more rickettsiae showing morphologic signs of destruction. Spheroplast-like forms occurred more often and were more numerous than in intact animals. Structures morphologically similar to L-forms of gram-negative bacteria and that of chlamydiae were also detected. After adding immune serum, more intact rickettsiae and spheroplasts were found in phagosomes as well as more phagolysosomes contained rickettsiae and spheroplasts with morphologic signs of destruction. It is suggested that clearance of immune macrophages from rickettsiae is mediated by at least two processes: on one hand by destruction of rod-shaped rickettsiae within phagolysosomes and, on the other hand, by formation and subsequent destruction of spheroplast-like forms within vacuoles, which probably also function as phagolysosomes.

The possibility of using IRMA in the authors' modification for the determination of rickettsiae and their antigens in different biological substances has been studied. The method has proved to be highly sensitive in the determination of the minimal amounts of antigens in diagnostic reagents and in rickettsial cell and egg cultures. The data obtained in this study indicate that there are good prospects for using this modification of IRMA in different specific investigations involving the detection of microorganisms and their antigens.

The possibility of L-transformation for two species of Rickettsia of the tick group (R. conorii and R. akari) was studied with light and electron microscopy. The experiments were performed on the cultures of L-cells (murine fibroblasts). Penicillin and vancomycin were used as the transforming agents. Electron microscopy revealed clear changes in the morphological structure of both species when the doses of penicillin and vancomycin were 50-100 and 250-1500 micrograms/ml, respectively. The antibiotics had a morphologically analogous effect on both species of Rickettsia. The resulting morphological structures were large oval spheroplast-like cells with significant expansion of the periplasmic space up to 1 X 1.75 micron under the effect of penicillin and 0.5 X 0.7 micron under the effect of vancomycin. R. akari was somewhat more sensitive to the antibiotics. Electron microscopy of the morphological changes in the Rickettsia induced by penicillin and vancomycin showed that they were similar to the changes observed in gram-negative bacteria on their L-transformation and to the penicillin forms of chlamydia (galprovia) and R. prowazekii. They may be considered as a phase of unbalanced growth and represent the initial stage of L-transformation.

Monolayer cultures of peritoneal macrophages of intact guinea pigs were infected with Rickettsia prowazekii (strain Breinl) and examined by electron microscopy after 30 min, 4 and 24 hr post-infection (p.i.). Three parallel processes developed in infected macrophages: reproduction of rickettsiae in macrophage cytoplasm, destruction in phagolysosomes and production of spheroplast-like forms. Reproduction of rickettsiae yielded 2 cell types: those with dense and with light cytoplasm; they were located side by side in the microcolony and seemed to have a common capsule-like coat. Relatively small spheroplast-like forms of about 1 micron in size were regularly detected. Addition of immune serum to macrophages increased the number of rickettsiae, both of rod-shaped as well as of spheroplast-like ones located within phagosomes, but elicited no increase in the number of digested pathogen cells.

Evidence has been obtained indicating the association of certain biological properties of Rickettsia prowazekii with their capacity for persistence. Highly virulent rickettsial cultures inducing increased levels of complement-fixing antibodies were shown to cause a high percentage of rickettsial carriers in infected cotton rats. Within an interval of 133-189 days after inoculation with the virulent Breinl strain, rickettsial carrier-state was still demonstrable in 20% of the animals. The vaccine strain E of R. prowazekii had a low capacity for persistence in cotton rats, because 64 days after inoculation all animals were found free of rickettsiae.

The ultrastructure of M. pneumoniae, grown on a solid culture medium and in a liquid one, was studied by a number of methods. Two types of cells were shown to prevail in the culture: spherical cells (0.5--1 micrometer) forming chains of different configurations and filamentous cells (5 micrometer long and greater) with spherical enlargements along their whole length. The absence of microcapsules made M. pneumoniae different from other species of mycoplasms, and the organism proliferated by division into 2 daughter cells, equal or unequal in size, by the segmentation of the cytoplasm and the formation of elementary bodies inside the cell and on its surface.

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.