[Southern taiga combined natural foci of spirochetoses].

AIM: Study of possibility of existence of combined natural foci of spirochetoses (ixodes tick borrelioses and leptospiroses) in typical taiga forests, and their etiologic and reservoir-host structure. MATERIALS AND METHODS: Small mammals of 19 species were captured in 1992-2010 at a station in low-mountain southern taiga forests of Chusov area of Perm region. Borreliae were isolated by seeding urinary bladder or aural bioptates into BSK II medium, leptospirae--by seeding a suspension of kidney tissue into Vervoort-Wolf medium. 1350 animals were studied by seeding for borrelia infection and 1077--for leptospira. 287 of those, small animals of 6 species, were simultaneously studied for borrelia and leptospira infection. Borrelia isolates were identified by using PCR and restriction fragment length polymorphism methods, and leptospirae--by using standard diagnostic agglutinating sera kit. Blood of 2893 rodents of 12 species and insectivorous of 7 species was studied in microagglutination reaction for the detection of antibodies against leptospirae. RESULTS: Infection by Borrelia garinii and Borrelia afzelii or Grippotyphosa serogroup leptospira was detected in 6 most numerous species of forest small mammals. 3 root voles and I bankvole were simultaneously infected by borreliae and leptospirae. B. garinii and Grippotyphosa serogroup leptospira were simultaneously isolated from 2 root voles, and B. garinii and Javanica serogroup Leptospira interrogans--from 1 root vole. A bank vole was infected by B. afzelii and Javanica serogroup leptospira. Mixed-infected animals composed 1.4% of all animals of background species studied in parallel. CONCLUSION: The data obtained indicate a presence of natural foci of leptospiroses in the southern taiga forest pre-Urals. The data confirm the conceptions regarding a predominant presence in European forest ecosystems of foci with Grippotyphosa serogroup L. interrogans pathogen, and the main carrier ofthese leptospirae being bank vole. Combined natural foci of spirochetoses of two groups (ixodes tick borrelioses and leptospiroses) were detected.

[Detection of leptospirosis infection in certain wild and domestic animals in Mongolia].

AIM: Serological examination for leptospirosis of domestic and certain species of wild animals in Mongolia. MATERIALS AND METHODS: Collection of material from domestic and wild animals was performed in 2009--2010 in 7 aimags (regions) of Eastern, Central and Southern Mongolia. Serological study of filter paper dried blood samples obtained from 51 specimens of cattle and small cattle, camels, and 545 specimens of rodents of various species was performed in microagglutination reaction (MAR) of leptospirae with 13 reference strains. RESULTS: There is a presence in certain regions of Mongolia of anthropurgic loci of leptospirosis infection including arid zones where ecological conditions do not favor the development of epizootic process. The results of the study indicate the epizootic significance of Tarassovi serogroup leptospirae in cattle and Sejroe serogroup (probably hardjo serovar) in goats, sheep and camels. Results of serological studies of desert and steppe specimens of wild fauna of Mongolia suggest a possibility of circulation of leptospirae in natural foci. CONCLUSION: Detection in a significant percent of cases in tarbagan and long tailed ground squirrel blood sera of agglutinins to Pomona (mozdok) leptospirae with negative MAR results for Pomona (pomona) strain suggests a presence of a pathogen of a previously unknown serovar. However final conclusion could be made only after the isolation of cultures of the pathogen and their identification.

[Banks of biological resources in the system of basic epidemiological and clinical studies].

Banks of biological resources appear to become the key centres of long-standing international scientific infrastructure necessary for efficacious use of achievements in public health. Approaches to building up the global system of monitoring socially significant and other dangerous infections based on the passported blood sera bank and computerized epidemiological database meeting the current WHO standards are discussed. An innovative project for the creation of the Electronic Atlas of Russia is considered that must provide an original information-analytical system for the study of the most widespread infectious diseases with the use of modern geoinformation technologies.

[Feasibility to use of recombinant domains of immunoglobulin-like protein LigA as a promising marker for serological testing of patients with leptospirosis].

AIM: To clone the DNA fragment encoding conservative domain of LigA protein of Leptospira interrogans into Escherichia coli and to investigate antigenic properties of constructed chimeric protein. MATERIALS AND METHODS: E. coli strain M15 [pREP4], recombinant plasmid pTT10 encoding cellulose-binding domain (CBD), restriction endonucleases BamHI, BglI, BglII, XbaI, T4 DNA-ligase, RNAse were used in the study. Molecular cloning of ligA gene fragment was performed using standard protocols, and expression of hybrid genes--according to "Qiagen company's protocols. Extraction and purification of proteins were performed using original method. RESULTS: DNA fragment encoding immunoglobulin-like domain 5 of LigA was cloned in E. coli. Effective strain-producer of chimeric domain D5-CBD consisting of the immunoglobulin-like domain 5 of LigA, Gly-Ser spacer, and cellulose-binding domain (CBD) was obtained. The high-purity D5-CBD preparation was obtained using one-stage purification on cellulose. Antigenic specificity of this chimeric protein was studied and it was shown that it could be used as a marker for the development of diagnostic ELISA kit. CONCLUSION: Recombinant domain of LigA in chimeric protein produced in E. coli retains antigenic properties of native LigA protein. Obtained results confirm the feasibility to use recombinant antigen D5-CBD as a marker for development of diagnostic kits on the basis of ELISA.

[Research into the immunogenic properties of the recombinant cellulose-binding domain of Anaerocellum thermophilum in vivo].

Development of new technology allows different antigens of a necessary degree of cleanliness to be obtained. This development is a major problem of modern medical biotechnology. A promising approach to this problem includes use of the affinity domains (tags) incorporated in structure of a recombinant antigen and capable to bind to corresponding sorbents. The method of preparation of ready-for-use injections containing complexes formed by soluble antigens on insoluble cellulose immunosorbent (not chemical conjugates) in one stage is based on the fusion protein technology. This approach includes preparation of two-component recombinant proteins containing an antigen of interest and the cellulose-binding domain (CBD), which spontaneously binds to cellulose containing sorbents with high binding constant. Research into the immunogenic properties of the CBD in the complex with cellulose and in the preparation of recombinant CBD in a rat model was performed. The titers of specific antibodies in rat serum induced by recombinant CBD and CBD in the complex with cellulose was evaluated. The CBD in the complex with cellulose was more immunogenic in comparison with CBD alone. The spectrum and levels of cytokines in collected rat serum induced by developed preparations was also measured using the microsphere-based Luminex Flowmetrix system (BioPlex). It was found that the amorphous cellulose was not an immunotolerant sorbent, because it induced the expression of the proinfammatory cytokines in vivo.

[The gene encoding the outer membrane lipoprotein lipL32 as a genetic target for developing leptospira genotyping schemes].

The primers flanking the fragments sized 677 bp (external) and 204 (internal) were constructed on the basis of nucleotide sequences of the gene encoding the outer membrane lipoprotein LipL32. PCR-analysis was used to study the prevalence of the gene lipL32 among 79 Leptospiraceae family strains representing different genera and genomic species (77--genus Leptospira, 1--genus Leptonema, 1--genus Turneria). The two amplicons were detected in the pathogenic leptospires--L. interrogans (except L. inadai), but not in saprophytic--L. biflexa. In L. inadai only 204 bp-amplicon was detected. These test-systems can be successfully used to differentiate between two distinct ecological groups of leptospires. The gene encoding the lipL32 seems to be appropriate as an adequate genetic target for developing the leptospira genotyping systems (high prevalence, presence of both conservative and variable sites in its nucleotide schemes).

[Prevalence of the gene encoding the outer membrane lipoprotein LipL32 in leptospires of different taxons].

Primers flanking the fragment sized 677 bp have been constructed on the basis of nucleotide sequences of the gene encoding the outer membrane lipoprotein LipL32. PCR-analysis was used to reveal the prevalence of gene lipL32 among 73 Leptospiraceae family strains representing different genera and genomic species. The gene lipL32 appeared to be conservative across the pathogenic species. In contrast, it was not detected in the genome of nonpathogenic free-living leptospires. Thus the developed PCR test-system with primers LEP21/LEP22 may be efficiently used to differentiate these two distinct ecological groups of leptospires.

[Evaluation of the epidemiological situation in different territories of the Russian Federation, based on the Bank of Standard Blood Sera].

The Bank of Standard Sera created in the Gamaleya Research Institute of Epidemiology and Microbiology provide for evaluation the epidemiological situation in any separate territory and in the whole of the country. The collection of certified blood sera is divided according to different territories of the country and makes it possible to obtain the retrospective and operative information on the level of population immunity and the immune structure of the population with respect to different infective agents, and to reveal the susceptible group of the population. The analysis of the unfavorable character of the epidemiological situation in the inspected territory with due consideration of its climato-geographical and anthropogenic environmental, socio-demographic characteristics, the level of population immunity and the immune structure of the population with respect to different infective agents makes it possible to carry out the epidemiological diagnostic (an outbreak, the import of infection, the use of a bacteriological weapon), prognosis and the prophylaxis of diseases, as well as the epidemiological cartography of territory.

[A new methodological approach for leptospira persistence studies in case of mixed leptospirosis]. / Novyi metodicheskii pogkhod k izucheniiu leptospir pri smeshannoi leptospiroznoi infektsii.

A new methodical approach for Leptospira persistence studies in case of mixed leptospirosis, based on the use of PCR test systems with different taxonomic specificity for the indication and identification of leptospires, was developed. Two PCR test systems (G and B) were used in experiments on BALB/c white mice to study patterns of the development of mixed infection caused by leptospires of serovar poi (genomospecies L. borgpeterseni) and grippotyphosa (genomospecies L. kirschneri). The conclusion was made of good prospects of this method application in studies on symbiotic relationships of leptospires both in vivo and in vitro.

[Bacterial zoonoses with natural focality: current trends in epidemic manifestation]. / Prirodnoochagovye bakterial'nye zoonozy: sovremennye tendentsii épidemicheskogo proiavleniia.

Current trends in epidemic manifestation of some bacterial zoonoses with natural focality and their role in human infectious pathology have been reviewed and analyzed. Update information on the etiological agents of "emerging" and "re-emerging" infections--Astrakhan spotted fever, bartonellosis, ixodes tick-borne borrelioses, monocytic erhrlichiosis and canine brucellosis, recently isolated on the territory of Russia, is presented. The main factors at play in the process of urbanization of bacterial zoonoses are discussed.

[Gene diagnostics of acute persistent Leptospira infection]. / Genodiagnostika ostroi i persistentnoi leptospiroznoi infektsii.

The results of clinical trials carried out in different foci have demonstrated high diagnostic value of analysis made with the use of the polymerase chain reaction (PCR) at early stages of Leptospira infection caused by infective agents of the serogroups Grippotyphosa, Canicola and Icterohaemorrhagiae. The possibility of leptospiremia lasting considerably longer than heretofore believed to be possible, as well as the persistence of leptospires in the liquor of patients after the acute phase of the disease is over, i.e. during the early and late convalescence periods, has been shown. This is indicative of good prospects of using the PCR analysis not only for early rapid diagnostics of Leptospira infections, but also for controlling the course of the infection, for prognosticating early and late complications of the disease, as well the mechanisms of pathogenesis.

[The evaluation of the reactogenicity and immunogenic activity of a new concentrated inactivated leptospirosis vaccine]. / Otsenka reaktogennosti i immunogennoi aktivnosti novoi kontsentrirovannoi inaktivirovannoi leptospiroznoi vaktsiny.

In the controlled field trial the reactogenicity, safety and antigenic activity of a new concentrated inactivated leptospirosis vaccine after its administration in one and two injections of 0.5 ml were studied in comparison with those of the existing commercial vaccine, introduced in two injections in doses of 2.0 and 2.5 ml. The new experimental vaccine exhibited low reactogenicity and was found to be safe and highly immunogenic when introduced in a single injection of 0.5 ml. As shown in this trial, the immunogenic characteristics of immunization made in a single injection were not inferior than those obtained as the result of immunization made in two injections, yielding high percentage of seroconversions (89.8% to 98.3%) with respect to 4 Leptospira serogroups and leading to the production of the protective titers of corresponding antibodies. The existing commercial vaccine was inferior to the experimental one in antigenic activity (the frequency of seroconversions, antibody titers). The results of the trial make it possible to recommend the experimental concentrated leptospirosis vaccine for use in medical practice in a dose of 0.5 ml introduced in a single injection.

[Analysis of genomic polymorphism in leptospira by polymerase chain reaction with random primers]. / Analiz genomnogo polimorfizma leptospir v polimeraznoi tsepnoi reaktsii s proizvol'nymi praimerami.

A test system for genetic typing of Leptospirae is developed, based on the polymerase chain reaction (PCR) with arbitrary primers. Thirteen strains of 4 Leptospira species were examined: L. interrogans, L. parva, L. illini, and L. inadai. Analysis of polymorphism of amplicon length (PAL) by the PCR with short Sh1 and Sh2 primers revealed genotypical differences at the inter- and intraspecias levels, as well as at the subserovar level. PAL of L. interrogans strains Rga and M-20, serovars icterohaemorrhagiae and copenhageni, were identical both with Sh1 and Sh2 primers. Moreover, PCR with Sh2 primer showed genotypical similarity between strains Moscow V and M. oeconomus, serovar grippotyphosa of the same serogroup. Analysis of PAL by the PCR with long Lgn1 and Lgn2 primers showed similar results. Analysis of the PAL values obtained by the PCR with all primers permitted us to differentiate 9 L. interrogans strains into 8 PAL genotypes and identify a different degree of genotypical relation between strains of different serovars of this species. Complete genotypical relationship between strains L. inadai N 10 and EMJH 86 (serovar lyme) was confirmed by the new test system. Therefore, PCR-based test system with different primers can be used to differentiate between closely related Leptospira strains and to investigate the genotypical relationships between the strains at the intra- and interspecies and inter- and subserovar levels.

[Development of polymerase chain reaction-based test systems for detecting leptospira in polytypical leptospirosis foci]. / Razrabotka test-sistemy na osnove polimeraznoi tsepnoi reaktsii dlia indikatsii leptospir v politipichnykh ochagakh leptospirozov.

Two highly sensitive test systems G and B, based on the polymerase chain reaction, were developed for indication of pathogenic Leptospira interrogans, including the serovariants appearing during outbreaks in polytypical foci of leptospirosis in the tropical zone of China. These test systems can be used for rapid diagnosis of leptospirosis in humans in foci with different etiological structure.

[The molecular genetic and biological aspects of the ecology of pathogenic Leptospira (Leptospira interrogans)]. / Molekuliarno-geneticheskie i biologicheskie aspekty ékologii patogennykh leptospir (Leptospira interrogans).

The results of recent investigations on the application of some genotyping methods (genomic fingerprinting, analysis of restriction length fragment polymorphism of DNA, etc.), made in order to study the population structure of pathogenic leptospires and to evaluate their intraspecies heterogeneity with regard to their main ecological features, are reviewed. New data on the use of PRC-based amplification test systems for the study of specific features of host persistence of leptospires are presented. The relative role of the parasitic and saprophytic phases of existence for populations of pathogenic leptospires belonging to different intraspecies taxa is discussed.

[A polymerase chain reaction method for studying host persistence of pathogenic leptospira]. / Metod polimeraznoi tsepnoi reaktsii v izuchenii gostal'noi persistentsii patogennykh leptospir.

Polymerase chain reaction has for the first time been shown to be applicable to indication of Leptospira interrogans in the organs of infected animals with acute or chronic leptospirosis (on the model of golden syrian hamsters). Polymerase chain reaction is superior to microscopic and bacteriological analyses in identification of leptospirae in organ suspensions. The sensitivity of the technique is 1-10 cells per sample in studies of kidney or brain suspensions or 100-1000 cells in studies of liver suspensions.

[Development of a test system for detecting Leptospira interrogans using the polymerase chain reaction]. / Razrabotka test-sistemy dlia vyiavleniia Leptospira interrogans metodom polimeraznoi tsepnoi reaktsii.

Based on polymerase chain reaction a test-system has been elaborated permitting one to identify the leptospirae of the most common serogroups (Icterohaemorrhagiae, Canicola, Javanica, Ballum, Pyrogenes, Pomona, Habdomadis, Sejroe, Tarassovi) of the species Leptospira interrogans. Sensitivity of the technique is 1-10 cells in a sample. The specificity of the system has been shown to depend on the temperature of the primers annealing. The elaborated system exceeds all other systems for leptospiral identification in sensitivity. It is prospective for leptospiral identification in biological liquids aimed at early diagnosis of leptospiroses and in the studies of leptospiral persistence in host organisms in the saprophitic phase of life cycle.

[A small-mammal study in the northeastern Kara Kum for leptospirosis infectivity]. / Issledovanie melkikh mlekopitaiushchikh severo-vostochnykh Karakumov na zarazhennost' leptospirozom.

Search for natural foci of leptospirosis was carried out in 1987-1989 in humid biotopes of Tashauz Province, Turkmenistan. Such potential carriers of leptospirosis as house mice (Mus musculus) and tamarisk gerbils (Meriones tamariscinus) are widely spread in this area, and the size of their population can amount to great numbers. The sharpest fluctuations in the population size are characteristic of house mice inhabiting the shores of water collecting ponds and lakes in the regions of minimally used pastures. A moderate number of house mice was registered in spring and autumn at the area of irrigated agriculture. Only here and only in autumn a faint epizootic manifestation of the natural focus of L. grippotyphosa infection in house mice was registered for the first time in Turkmenistan. The titers in mouse blood sera, determined in the microagglutination test and the Leptospira lysis test, were 1:20 to 1:200. According to the data on the humidity and pH of the soil in the vicinity of irrigated fields, spring and summer months may be regarded as the most favorable period for the survival of leptospires in the environment. The probability of the aggravation of the epizootic situation seems to increase when irrigated fields adjoin pastures of are temporarily used as grazing ground for cattle.