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Aim: An attempt to use MALDI-TOF mass-spectrometry method for identification of lep- tospiral isolates on the serovar level., Materials and Methods: 8 reference Leptospira spp. and 11 leptospira strains isolated from leptospiral patients and infected animals in the North-Western region of Russia were included into the study. Mass-spectra of all the studied strains were obtained by direct profiling of cell extracts. The created main spectral profiles (MSP) of reference strains were used for identification of isolates. Evaluation of identification was carried out by calculating coefficients of matching rate of separate spectra of each isolate with MSP of all the reference strains., Results: Results of identification have shown the similarity of spectra of isolates belonging to Pomona, Icterohaemorrhagiae and Canicola serogroups, with MSP of saprophyte strain L. biflexa Patoc I. It is assumed that spectra of the studied strains contained peaks of polysaccha- ride Ο-antigens. Wherein maximum mean values of matching rate coefficients between spectra of isolates and MSP of pathogenic reference strains of leptospira correctly matched serovar type of the isolate., Conclusion: Further extended studies may form the base of development of a simple and rapid method oftyping of leptospirosis causative agents on the level of serovars using MALDITOF mass-spectrometry.

Aim: Comparison of diagnostic capabilities of 2 variants of PCR for detection of Coxiella burneli persistence in dynamics of infectious process in patients with Q fever., Materials and Methods: 110 samples of clinical material, obtained from patients with Q fever in an endemic region for this infection (Astrakhan region), were studied. The samples were studied in a standard PCR (marker - 16S rRNA gene fragment) and in real-time. PCR (RT-PCR) (marker - groEL gene fragment)., Results: Both markers were established to be perspective for detection of C. burnetii DNA in clinical material, and RT-PCR detects positive result including late stages of the disease (illness day 21 - 31)., Conclusion: This study is the first Russian publication on comparison on different PCR variants for detection of C. burnetii in blood of Q fever patients in dynamics of the infectious process.

Aim: Comparative typing of Leptospira spp. strain collection based on analysis of 16S RNA fragment., Materials and Methods: 2 pairs of primers were used for PCR, that jointly flank 1423b.p. sized fragment. Sequences of Leptospira spp. strain 16S rRNA, presented in the international database, were used for phylogenetic analysis., Results: A high similarity, including interspecies, of the 16S fragment in Leptospira spp. strains was shown independently of the source, serovar and serogroup. Heterogeneity of the primary matrix, spontaneous mutations of hotspots and erroneous nucleotide couplings, characteristic for 16S sequence of pathogenic Leptospira spp. strains, are discussed. Molecular-genetic characteristic of certain reference Leptospira spp. strains by 16S sequence is obtained., Conclusion: Results of the studies give evidence on expedience of introduction into clinical practice of identification of Leptospira spp. by 16S sequence directly from the clinical material, that would allow to significantly reduce identification time, dismiss complex type-specific sera and other labor-intensive methods.

Aim: Study of the ability of clinical isolates of leptospira to cause production of certain pro- and antiinflammatory cytokines in the model of human whole blood., Materials and Methods: Leptospira interrogans strain was taken for the experiment. Cytokine content was determined by a method based on xMAP technology using a standard panel, composed of 9 analytes: TNF-α, MCP-1, IL-8, IL-4, IL-6, IL-10, IL-IRa, IL- 12 (p70), IFN-γ., Results: An optimal concentration of L. interrogans was selected for stimulation of human whole blood--1 x 10(6) leptospirae/ml. For the first time in the model of human whole blood it was determined, that at early stages of incubation IFN-γ, IL-12(p70), IL-4 and IL-1Ra are more actively produced; at later stages (6 hour incubation)--IL-8 and TNF-α., Conclusion: A differential pattern of cytokine production stimulation was shown in the model of human whole blood by live and inactivated leptospirae.

Aim: Creation of a classification model of Leptospira spp. serovar model using ClinProTools 3.0 software and evaluation of use of MALDI-TOF MS as a method of quality control of reference strains of leptospira., Materials and Methods: 10 reference strains of Leptospira spp. were used in the study according to microscopic agglutination reaction from the collection of Pasteur RIEM. All the strains were cultivated for 10 days in Terskikh medium at 28 degrees C. Cell extracts were obtained by ethanol/formic acid method. α-cyano-4-hydroxycinnamic acid solution was used as a matrix. Mass-spectra were obtained in Microflex mass-spectrometer (Bruker Daltonics, Germany). External validation of the test-model was carried out using novel spectra of every reference strain during their repeated reseeding., Results: Values of cross-validation and confirmatory ability of the optimal model, built on a genetic algorithm, was 99.14 and 100%, respectively. This model contained 11 biomarker peaks (m/z 2959, 3447, 3548, 3764, 3895, 5221, 5917, 6173, 6701, 7013, 8364) for serovar classification. Results of the external validation have shown a 100% correct classification in serovar classesin Sejroe, Ballum, Tarassovi; Copenhageni, Mozdoc, Grippotyphosa and Patoc, that indicates a high prognostic ability of the model in these classes. However, data from verification matrix have shown, that 50%.of the spectra from Canicola and Pomona serovars were classified as Patoc class, that could be associated with cross serological activity of Patoc serovar L. biflexa with pathogenic leptospirae., Conclusion: MALDI-TOF mass-spectrometry method combined with building and using the classification model could be a useful instrument for intra-laboratory control of leptospira reseeding.

Aim: Study the content of some pro- and anti-inflammatory cytokines in blood sera of leptospirosis patients in dynamics of infectious process and the role of these cytokines in the disease immunopathogenesis., Materials and Methods: The content of cytokines in blood sera was determined by a method based on xMAP technology with a standard panel consisting of 9 analytes: TNF-α, MCP-1, IL-8, IL-4, IL-6, IL-10, IL-1Ra, IL-12 (p70), IFN-γ., Results: A significantly increased level of IL-8, IL-10, TNF-α was confirmed and the increased content of MCP-1 in leptospirosis patients compared with practically healthy donors was established for the first time. Correlations between cytokines during leptospirosis were detected., Conclusion: The data obtained show that cytokines play an important role in leptospirosis immunopathogenesis.

Aim: Development of primers for the detection of leptospirae in clinical material including urine., Materials and Methods: Study of specificity and sensitivity of primers complementary to colA gene in standard PCR by using DNA preparation of cultures of pathogenic and saprophytic leptospirae, biological materials from healthy humans and dogs, including contaminated with pathogenic leptospirae culture., Results: Specific interaction of these primers with DNA of pathogenic leptospirae of 14 serogroups was established. Sensitivity of the technique was 50 cells in 1 ml of sample., Conclusion: The primers described fulfill the requirements for the sensitivity and specificity and can be recommended for the detection of leptospirae in both serum and urine.

We analyzed restriction fragment length polymorphism of lipL32 gene encoding outer membrane protein in three Leptospira genomic species (L. interrogans, L. kirschneri, and L. borgpetersenii) using Bsa29 I and Bam HI restriction endonucleases. It was found that restriction profiles of the studied gene were similar at both the intraspecific and serogroup levels; variability was revealed only at the interspecific level, which can be explained by relatively low variability of lipL32 gene.

Aim: Improvement of PCR for detection of Coxiella burnetii in field material by development of set of primers for amplification of groEL gene fragment., Materials and Methods: C. burnetii strains, samples from organs of wild rodents and laboratory animals, blood of healthy donors and laboratory animals. Methods of DNA isolation, PCR, and electrophoresis in agarose gel were used., Results: Nucleotide sequences of primers amplifying groEL gene fragment, which could increase specificity of PCR during work with field material were proposed., Conclusion: Obtained data open perspective for using this method for detection of C. burnetii in environment.

Short information about significance of Q-fever in human pathology is represented. Necessity of vaccination is proved. The row of vaccines, developed in Czechoslovakia and Romania and identified as small-effective, was considered. Live vaccine from M-44 strain, was made in USSR, still remains in Russia. However, experimental data of American and Russian authors showed persistency of Q-fever agents in vaccinated animals, abortions and other pathology. WHO recommended declining to use live vaccines. Inactivated corpuscular combined vaccine against Q-fever was development under leadership of I. V. Tarasevich. The method of vaccine production is protected by industrial patent #2094057 from 31.01.94, concomitant studies--by 9 author's certificates. The vaccine is harmless, are actogenic, and high immunogenic after single injection. Antibodies of vaccinated persons remain more than in 75% during one year. The vaccine assists in resolving of actual problems of fight against Q-fever.

The author describes cases of secondary leptospirosis. Secondary infection with leptospiras occurred in patients with concomitant pathology. In connection with occupational activity or life-style the patients got infected from Rattus norvegicus.

A study of possible transovarial and transphase transmission of borreliae by the tick Dermacentor reticulatus is carried out. The possibility of borreliae transmission by the ticks of this species being infected spontaneously as well as experimentally is shown in principle. Although borreliae are preserved in the organism of D. reticulatus, low values of the infestation are established in the different stages of D. reticulatus development and in all steps of the study. D. reticulatus may be involved in the process of borreliae circulation in natural foci of tick-born borreliosis, but this species has no a significant importance for the maintenance of it.

The study of immune response in Q-fever patients and convalescents at different periods from the beginning of the disease demonstrated the diagnostic possibilities of the complement fixation test (CFT), the microagglutination test (MAT), the passive immunofluorescence test (PIFT) and the enzyme immunoassay (EIA). High effectiveness of EIA in comparison with CFT, MAT and PIFT was noted (97, 63, 87 and 91% respectively). The sensitivity of EIA was not less than 100-250 times higher than that of CFT, 15-100 times higher than that of MAT and 30-80 times higher than that of PIFT.

In this work the evaluation of macro- and micro-changes in the tissues of guinea pigs to the injection of inactivated antigens of C. burnetii is presented. The injection of these antigens in a dose of 50 micrograms induced only an insignificant reaction at the site of injection; in the brain of the animals, as well as in all examined internal organs, no visible pathological changes were present. During the first two weeks after the injection of the antigens signs of irritation of the reticuloendothelial system and hyperplasia of lymphoid tissue in T- and B-dependent zones were observed in the regional lymph nodes of the experimental animals. These changes became moderate by day 28.

The cultivation of mouse peritoneal macrophages in the presence of antigenic preparations obtained from C.burnetii was accompanied by the appearance of phagocytes and considerable amounts of tumor necrosis factor and interleukin 1 in the culture medium. The production of cytokines depended on the doses of preparations used as inducers. The special treatment of C.burnetii antigen, selectively removing its phospholipid components, led to a sharp drop in its capacity for stimulating the production of cytokines.

The immunogenicity of the Coxiella burnetii corpuscle antigen and two its components, obtained after the treatment with chloroform-methanol or extraction by trichloroacetic acid, was investigated. All the studied antigens were highly immunogenic. A strong correlation was discovered between the immunogenic and immunomodulating properties of the C. burnetii antigens. Cells of the mononuclear phagocytic system are the targets for stimulating action of the studied antigens. A cyclic increase of antibacterial activity and oxygen-dependent metabolism of the peritoneal cells was noted, in addition to the increase of the natural splenocyte cytotoxicity caused by the action of these antigens. The role of cytokines in rising the activity of peritoneal cells and splenocytes, caused by immunization of mice with the above antigenic substances, is discussed.

It was shown that the immunization of mice by the chemical Q fever vaccine prepared with the use of the strain "Coxiella burnetii, Nine Mile", phase I did not markedly activate the oxidative metabolism of the peritoneal exudate cells and did not induce the non-specific cytotoxicity in them. However, it resulted in a significant suppression of the mitogen-induced proliferation of splenocytes on days 3-5. The immunization of mice by the combined (corpuscular/chemical) vaccine prepared with the use of the strain "Apodemus flavicollis-Luga", phase I induced a significant activation of the oxidative metabolism of the peritoneal exudate cells and the development of the nonspecific cytotoxicity in them, whereas no suppression of the mitogen-induced proliferation of splenocytes was observed. The differences are likely associated with the presence of the corpuscular component in the combined vaccine and the strain antigenic specificity.

The structural heterogeneity in Coxiella burnetii chromosomal DNA isolated in the European part of Russia from people, agricultural animals, and ticks has been studied. It is compared with the one of the European strains Henzerling and M44, the only genetically characterized strains up to date. The digestion of the total DNA by the restriction endonucleases BamHI, PstI, XhoI resulted in obtaining two types of restriction patterns. The ones for Henzerling and M44 differed from the restriction patterns of the Russian strains, while the latter proved to be identical. The obtained data are in proof of genetical homogeneity in the Russian group of strains. The group is different from the genomic group including Henzerling and M44. The fact is in proof of the genetical heterogeneity of the European population of coxiellae.

Blood of 56 guinea pigs with experimental Q rickettsiosis was studied cytochemically (lysosomal cationic test) to measure the level of cationic proteins in neutrophil granulocytes. Development of Q rickettsiosis resulted in a decrease in the killing ability of neutrophils, depending on infection dose introduced. However, by day 7 of the disease, the level of cationic proteins in blood neutrophil granulocytes returned to the initial range. Similar situation was noted after subcutaneous injection of Coxiella burnetti corpuscular antigen. Subcutaneous infection with the living culture stimulus induced the wave-like decrease of the cationic proteins content. Infection of pre-immunized animals led to smaller decrease in the cationic proteins levels and to their more rapid recovery. Aspects of antimicrobial activity of neutrophil granulocyte cationic proteins in experimental Q rickettsiosis is discussed.

Preliminary injections of human recombinant tumor necrosis factor (TNF-alpha) and interleukin 1 (IL-1 beta) increases the resistance of guinea pigs to Coxiella burnetii. The increase of this resistance depends on the dose of cytokines and the time of their inoculation.

After the intraperitoneal injection of corpuscles of C. burnetii antigen (Ag), phospholipid (PL), and sediment obtained after the extraction of PL from Ag with chloroform-methanol (CM) slight leukocytic reaction developed in the peritoneum on day 1, and on day 2 it could be observed in the liver and in the spleen. Ag induced the most pronounced morphological changes. In the spleen they were manifested by the activation of T- and B-dependent zones of white pulp from day 2 and by the pronounced hyperplasia of reticular cells and macrophages, leading to splenomegaly, by days 7-14. Simultaneously lymphoid-macrophagal granulomas and hepatomegaly developed in the liver. By days 7-14 the foci of necrosis in the liver were caused by the thrombosis of portal veins and were not registered after the injection of PL and CM (and earlier also in experiments with Ag in doses of 0.1-0.3 mg).

Materials on the development of an enzyme immunoassay (EIA) system for the detection of the antigens of C. burnetii, the causative agent of Q rickettsiosis, are presented. The system is highly specific and effective with respect to both corpuscular antigens of phases 1 and 2 and soluble antigen (lipopolysaccharide). The sensitivity of this method varies within the range 5-100 ng/ml. The effectiveness of EIA as a quantitative (semiquantitative) control test used in the process of the production of Coxiella preparations has been demonstrated.

It is proved that the challenging of the extract isolated from Coxiella (as a result of treatment with the mixture of chloroform and methanol) to animals doesn't provoke the appearance of antibodies to these microorganisms. It also doesn't cause the sensibilization of lymphocytes to Coxiella, but the extract can interact specifically with a serum containing antibodies to Coxiella. It permits to characterise the tested extract of Coxiella as a hapten with properties of adjuvant because it stimulates antibody-appearance.

In one of the rural regions of the nonchernozem zone retrospective epidemiological diagnosis was carried out in connection with high morbidity rates in acute respiratory diseases and influenza. This diagnosis was shown to cover cases of Q fever. Altogether 172 cases of this infection were retrospectively diagnosed. This infection affected persons working at one of sheep-breeding complexes, as well as at enterprises, technologically linked with this complex. Sheep proved to be the source of this infection, that was confirmed both serologically and by the registered cases of the isolation of Coxiella strain from sheep.

The evidence of the prolonged persistence of Coxiella burnetii in experimental animals was obtained. Cyclophosphamide was shown to be capable of activating Q rickettsiosis in guinea pigs, and the period of the aggravation of this infection was found to be accompanied by a decrease in IgG concentration and a relative increase in IgM content.

The combined use of the complement fixation test, the indirect immunofluorescent test and the ring precipitation test with C. burnetii antigen greatly enhances the effectiveness or serological study, as it allows not only to find out the spread of infection among population more completely, but also to differentiate, to a certain extent, "fresh" infectious process from immunological trace reaction. The arguments are presented in favor of introducing the antigen of C. burnetii, phase I, into practice, especially in surveying the sera of farm animals.

Materials are submitted on electron-microscopic studying of alive and inactivated Coxiella in their interaction with murine cells during 28 days observations. The most variability of Coxiella morphologic types is shown to be found in spleen. The structure of endospora type is described. Several types of phagosomes are revealed and described.

The data on the epidemiological analysis of 675 cases of infection of the typhus nature, detected in Leningrad in 1974-1986, are presented. The seasonal morbidity curve, the age structure of patients, the character of the clinical course of the disease and other data gave a reason for the diagnosis of Brill's disease. The comparison of the relative data on morbidity rates in typhus at different periods of the current century was made.

In trials the optimal conditions of immuno-enzyme analysis for detection of antibodies to Coxiella burnetii were revealed. It was proved that the sensitivity of ELISA exceeds the sensitivity of reaction of complement binding not less than in 100 times. The effectiveness of ELISA was shown to be higher than that of reaction of complement binding both at early terms of disease (up to 14th day) and in persons having the disease rather long ago (1-2 years ago).

Soluble antigen obtained by the treatment of Coxiella burneti of the I phase with trichloracetic acid could be used in the capacity of a precipitinogen in the band-precipitation reaction in the diagnosis of Q-rickettsiosis and in mass epidemiological studies.

A case was described when a newborn was inoculated with Coxiella Burnetti as a result of infection relapse (in 8 months after acute disease) in his mother. Coxiella Burnetii were isolated from blood of both patients. Dynamics of humoral immunity indices in mother was observed both at the primary disease and during the period of infection relapse.

A new method for the preparation of Q-fever erythrocyte antigenic diagnosticum, adaptable to large-scale production, has been developed, and the diagnosticum thus obtained has been found to be highly specific and sensitive. The combined use of the complement fixation and passive hemagglutination tests enhances the effectiveness of the serological study, as it not only ensures a more complete detection of antibodies to C. burnetii, phase I, in humans and cattle, but also gives more precise indications on the time of infection.