[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.

[MALDI-TOF mass-spectrometric analysis of cell proteins during identification of leptospira genus members].

AIM: Development of methodological approaches for identification of leptospira by using MALDI-TOF direct protein profiling technology. MATERIALS AND METHODS: Analysis of cell proteins of 34 leptospira strains was carried out in Microflex LT by using "MALDI Biotyper 3.0 for identification and classification of microorganisms" program. RESULTS: 19 reference spectra of reference leptospira strains from 7 species were generated and imported into MALDI Biotyper 3.0 database. Identification of 6 strains with undetermined taxonomic position was carried out. CONCLUSION: The approved method allows determination of leptospira species with accuracy that depends on their adaptation to nutrient media, preparation approach and sample storage conditions for mass-spectrometry.

[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.

[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.

[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.

[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.

[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.

[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.

Leptospires colonial variations.

Six of 12 relatively freshly isolated and museum strains representing serovars mozdok, monjakov, kazakhstanica I and patoc were heterogeneous in colonia morphology. Recloned colonial variants of one and the same heterogeneous population did not exhibit any differences in antigenic properties in cross reactions of microagglutination and absorption of agglutinins. Along with it among the clones with various colonial types of the two relatively freshly isolated strains of serovar mozdok distinctions in virulence (LD50) for hamsters were marked. Moreover for the clones one of them differences in the level of renal infection (ID50) and in morphology of the cells (hooked and straight) were found. In a solid Tween-80 albumin medium in populations of two colonial variants (serovars monjakov and kazakhstanica I) colonial mutants appeared without any changes in antigenic properties, virulence and cell morphology with frequency of 10-9-10-8 per one bacterium per one generation.

Potential variability of Leptospira serovars belonging to the same group.

3 clone strains of monjakov and pomona serovars were cultured with prolongation in a medium containing homologous immune sera. Separate subcultures were cloned in a solid medium, while isolated clones were typed in microagglutination test with whole and absorbed immune sera and in some cases in an absorption test. The results showed that the mutants of subcultures with the minimal and maximal time of contact with the immune serum turned out to differ greatly from each other in antigenic properties. While antigenic mutants of earlier subcultures were constantly connected with serovars of Pomona serogroup, mutants of subcultures adapted to immune serum were serologically more related to butembo serovar of Cynoptery serogroup. The results obtained showing potential variability of serovars belonging to Pomona serogroup could be significant from the viewpoint of classification and phylogeny of Leptospira.

[Sensitivity of certain strains of pathogenic Leptospira to streptomycin, the nature of resistant variants and the frequency of their occurrence]. / Chuvstvitel'nost' nekotorykh shtammov patogennykh leptospir k streptomitsinu, priroda ustoichivykh k nemu variantov i chastota ikh vozniknoveniia

Streptomycin sensitivity of 112 Leptospira strains isolated from domestic and wild animals was studied. Independent of the isolation period and nature the strains proved to be highly sensitive to the antiobiotic (MIC 0.1 to 0.5 gamma/ml). On the example of a clone strain of Leptospira it was shown with a fluctuation test that streptomycin resistance occurred spontaneously. Frequency of the streptomycin resistant mutations was determined for 5 clone strains of L. interrogans. It was 10-9 to 10-7 per a Leptospira.