Coxiella burnetii in ticks and wild birds.

The study objective was to get more information on C. burnetii prevalence in wild birds and ticks feeding on them, and the potentialities of the pathogen dissemination over Europe by both. MATERIALS: Blood, blood sera, feces of wild birds and ticks removed from those birds or from vegetation were studied at two sites in Russia: the Curonian Spit (site KK), and the vicinity of St. Petersburg (site SPb), and at two sites in Bulgaria: the Atanasovsko Lake (site AL), and the vicinity of Sofia (site SR). METHODS: C. burnetii DNA was detected in blood, feces, and ticks by PCR (polymerase chain reaction). All positive results were confirmed by Sanger's sequencing of 16SrRNA gene target fragments. The antibodies to C. burnetii in sera were detected by CFR (complement fixation reaction). RESULTS: Eleven of 55 bird species captured at KK site hosted Ixodes ricinus. C. burnetii DNA was detected in three I. ricinus nymphs removed from one bird (Erithacus rubecula), and in adult ticks flagged from vegetation: 0.7% I. persulcatus (site SPb), 0.9% I. ricinus (site KK), 1.0% D. reticulatus (AL site). C. burnetii DNA was also detected in 1.4% of bird blood samples at SPb site, and in 0.5% of those at AL site. Antibodies to C. burnetii were found in 8.1% of bird sera (site SPb). C. burnetii DNA was revealed in feces of birds: 0.6% at AL site, and 13.7% at SR site. CONCLUSIONS: Both molecular-genetic and immunological methods were applied to confirm the role of birds as a natural reservoir of C. burnetii. The places of wild bird stopover in Russia (Baltic region) and in Bulgaria (Atanasovsko Lake and Sofia region) proved to be natural foci of C. burnetii infection. Migratory birds are likely to act as efficient "vehicles" in dispersal of C. burnetii -infested ixodid ticks.

[IDENTIFICATION OF LEPTOSPIRA SEROVARS BY MALDI-TOF MASS-SPEC- TROMETRY].

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.

[TYPING OF LEPTOSPIRA SPP. STRAINS BASED ON 16S rRNA].

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.

[COMPARISON OF DIAGNOSTIC EFFECTIVENESS OF METHODS OF DETECTION OF COXIELLA BURNETII IN BLOOD OF PATIENTS WITH Q FEVER BASED ON AMPLIFI- CATION OF 16S rRNA GENE FRAGMENTS (STANDARD PCR) AND groEL GENE (REAL- TIME PCR)].

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.

[PRODUCTION OF CERTAIN PRO- AND ANTI-INFLAMMATORY CYTOKINES DURING STIMULATION BY LIVE AND INACTIVATED LEPTOSPIRA PATHOGENS IN THE MODEL OF HUMAN WHOLE BLOOD].

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.

[MALDI-TOF MASS-SPECTROMETRIC ANAIYSIS OF LEPTOSPIRA SPP. USED IN SERODIAGNOSTICS OF LEPTOSPIROSIS].

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.

[Content of some pro- and anti-inflammatory cytokines in blood sera of leptospirosis patients].

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.

[Development of PCR test system based on colA gene for detection of leptospirae in clinical material].

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.

Interspecific polymorphism of gene lipL32 restriction profiles of pathogenic leptospires.

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.

[Improvement of method of Coxiella burnetii detection in biological material based on groEL gene amplification].

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.

[RAMS academician I. V. Tarasevich is a leader of development of combined inactivated vaccine against Q-fever].

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.

Study of heterogeneity of Coxiella burnettii strains by analysis of groEL gene restriction fragment length polymorphism.

Restriction fragment length polymorphism was used for evaluation of genetic heterogeneity of Coxiella burnettii strains. A 594-b. p. fragment of groEL gene was cleaved with MN1I, HpaII, and Hin6I endonucleases. Genetic homogeneity of Coxiella burnettii strains from the Russian collection and their relation to the group of European and North American strains were detected.

[Secondary leptospirosis].

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.

Anthropogenic effects on changing Q fever epidemiology in Russia.

In the northwestern region of Russia (Leningrad province) cattle is proved to be the main source of C. burnetii infection in humans, both in menaced professionals and in formally nonmenaced groups. Liquidation of specialized cattle-breeding complexes (with their well-organized veterinary surveillance) and broadening of the circle of non-professionals that contact with agriculture or domestic animals infected with C. burnetii provide the prerequisites to Q fever spreading among various groups of population.

[A study of possible transovarial and transphase transmission of borreliae by the tick Dermacentor reticulatus (Ixodidae)].

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.

Detection of Coxiella burnetii by PCR in mice after administration of live M-44 vaccine.

Original primers were prepared for the detection of coxiella burnetii by PCR for the amplification of a 752-bp fragment of dnaJ gene. Using these primers we observed the persistence of C. burnetii in different organs of mice after the administration of live Q-fever vaccine over a 7-months period.

[The immune response in those ill with and convalescing from Q fever studied by using immunoenzyme analysis]. / Izuchenie immunnogo otveta u bol'nykh i perebolevshikh Ku-likhoradkoi pri ispol'zovanii immunofermentnogo analiza.

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.

[The morphological changes in the body of guinea pigs in response to the administration of different Coxiella burnetii antigens]. / Morfologicheskie izmeneniia v organizme morskikh svinok v otvet na vvedenie razlichnykh antigenov Coxiella burnetii.

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.

[Coxiella burnetii antigens--inducers of tumor necrosis factor and interleukin-1]. / Antigeny Coxiella burnetii--induktory faktora nekroza opukholi i interleikina-1.

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.