FIRST MOLECULAR IDENTIFICATION OF THE TULAREMIA AGENT IN THE TICKS IXODES TRIANGULICEPS BIR. IN RUSSIA.

The ticks Ixodes trianguliceps (140 nymph pool and 211 adults) collected from small forest mammals in the forests of the Middle Urals (Chusovskoy district of the Perm Region) were tested using real-time PCR for the presence of Francisella tularensis DNA. Using the target gene 16S rRNA, the locus size 1165-1170 bp Francisella DNA was detected in 12 adults and 4 pools of nymphs. DNA-positive samples from 17 individuals from 128 adults and in 16 of 89 nymph pools were additionally detected by amplification of a shorter locus of the same gene (221-222 bp). All 49 16S rRNA gene-positive samples of real-time Taqman PCR assays directed against the tul4 (lpnA) gene locus and ISFtu2 element were identified as F. tularensis. These data suggest the possible involvement of the ticks I. trianguliceps in the circulation of the causative agent of tularemia in the natural foci of the forest type.

[Parasitological factors impeding the transmission of the agent of babesiosis (Babesia microti) to man from the tick Ixodes persulcatus].

Based on the analysis of own and literature data, it is concluded that the following ma- in permanent system of ecologicalarasitological factors prevents the effective vector functions of the tick I. persulcatus in transmission of B. microti: lack of distinct nymphs' anthropophily; small spontaneous invasion of hungry adults; a duration of the parasitic phase in humans is insufficient to complete the sporogonic development, because victims interrupt the phase. Therefore, not excluding the possibility of sporadic babesiosis disea- ses, it can be stated that within the boundaries of a vast territory, where the taiga tick is the only potential source of infection for humans, the B. microti infection has not, and will not reach significant values in infectious pathology.

[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 natural tularemia foci in Mongolia].

AIM: Study of the current spread of natural tularemia foci in Mongolia and its epizootic activity evaluation for consequent substantiation of the recommendations for prophylaxis of this disease. MATERIALS AND METHODS: Study of 1119 pellet specimens from predatory birds obtained in 6 aimag in Mongolia in 2008--2010 was performed. Tularemia antigen was detected by using antibody neutralization reaction (ANR) and passive hemagglutination reaction (PHR) with tularemia diagnosticums. Tularemia DNA was detected by PCR by using strain specific primers. Presence of plague antigen in PHR with plague immunoglobulin diagnosticum was also studied in all the samples. RESULTS: Epizootologic monitoring allowed the detection of natural tularemia foci in 5 of the 6 studied aimags in Mongolia. PHR was the most effective study method that allowed to detect tularemia antigen in the environmental objects in high quantities (up to 9.2% of positive samples) and high titers (up to 1:1600). PCR was less effective. Plague antigen was detected in 9 samples in 2010 for the first time, and in 3 cases together with tularemia antigen, which indicates a presence of combined natural foci of tularemia and plague in this territory. CONCLUSION: In the studied regions of Mongolia natural tularemia foci were detected, their epizootic activity was determined and recommendations for future study tactics of natural tularemia foci were given.

[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.

[Etiological structure of complex Ixodes tickborne borreliosis natural foci of southern taiga].

89 primary isolates of B. garinii and 72 B. afzelii from different developmental phases of I. persulcatus, I. trianguliceps and form small mammalian hosts of Borrelia were obtained at an area of ca. 30 km2 located in low-mountain southern taiga forests (Perm region). The area provides home for two Borrelia species (B. garinii and B. afzeli) and their natural carrier Ixodes persulcatus. 23 isolate of B.garnii were obtained from skin biopsies and blood samples taken in patients with borreliosis. The isolates were studied by sequencing rrf(5S)-rrr(23S) spacer. The term genetic variant (genovariant) is proposed for the totality of isolates belonging to a given genetic subgroup of the concrete genospecies and having a similar nucleotide sequence of rrf(5S)-rrr(23S) spacer or other conservative genomic sequence. Genovariant is ths smallest intraspecies taxonomic unit in widespread Borrelia pathogenic for man. Several genovariants of B. garinii and B. afzelii may simultaneously occur in combined parasitic systems formed by these spirochetal agents of Ixodes tick-borne borreliosis. Such natural foci in southern taiga of the Perm region have a complicated etiological structure due to the presence of 14 genovariants of Borrelia belonging to the two above genetic subgroups. Specific genovariants occur annually but with different frequency. They are lacking in host-specificity.

[Genetic characterization of the Brucella melitensis isolates from Mongolia, Russia, and Azerbaijan].

UNLABELLED: The goal of this work was to provide comparative genetic characterization of the human and animal Brucella melitensis isolates from Mongolia, Russia and Azerbaijan using current molecular-genetic typing methods. MATERIALS AND METHODS: Twenty eight Mongolian (n = 18), Russian (n = 6), and Azerbaijan (n = 4) human and animal Brucella melitensis isolates were studied using 2 molecular typing methods based on PCR for differential species and biovar specific ORF (open reading frames) molecular targets and MLVA (multiple locus variable number tandem repeats analysis) using primers to 12 known loci. RESULTS: The PCR was used for differential molecular targets (all B. melitensis isolates) were characterized as the B. melitensis biovar 2. The MLVA revealed 7 identical and 5 variable MLVA loci. All the isolates were classified into 25 genotypes using the dendrogram on the data of 12 loci and the cluster related to reference strain B. melitensis 63/9 biovar 2. The B. melitensis isolates having related MLVA genotypes were connected to Mongolian, Russian and Azerbaijan regions. The circulation for two B. melitensis isolates to not typical hosts as camel and yak was demonstrated using molecular typing methods. CONCLUSION: The genetic characterization of twenty eight B. melitensis isolates from different geographical regions in Mongolia, Russia, and Azerbaijan recognized genetic relationships. On the other hand, the MLVA has high discrimination power with a resulting Hunter and Gaston discriminatory index (HGDI) of 0.9841 revealing genetic diversity for the isolates forming of 25 MLVA genotypes. To improve the system of the brucellosis surveillance in Russia MLVA typing of B. melitensis isolates are necessary to investigate from the Siberian (Republics Tuva, Buryatia, and Irkutsk region) and South (Republics Dagestan, Kalmykia, and Stavropol region) Districts having frontier areas with Mongolia and Azerbaijan.

[Genetic variants of Borrelia garinii, a widely spread Eurasian pathogen of ixodic tick borreliosis].

As a result of PCR-RFLP analysis and the degree of similarity between the nucleotide sequences analysis of the rrfA-rrlB intergenic spacer DNA of 227 primary isolates of Borrelia garinii and 71 isolates/ amplicons from GenBank database in different regions of Eurasia revealed significant intraspecific heterogeneity among those of Borrelia. It was shown that genospecies B. garinii had within the two genetic subgroups (20047 and NT29) 16 genetic variants, whose geography was likely to be different.

[Molecular genetics typing of Brucella circulating in several provinces of Mongolia].

AIM: Comparative molecular-genetic typing of Brucella strains isolated in Mongolia from different animal species as well as from humans. MATERIALS AND METHODS: Twenty-one strains of Brucella isolated from different hosts in 7 provinces of Mongolia were typed. Conventional phenotypic methods, genotyping by PCR with primers for genus- and species-specific differentiating targets of Brucella genes as well as multiple locus variable number tandem repeats analysis (MLVA) with 12 pairs of primers bounding locus variable tandem repeats of different length (from 134 bp to 8 bp). RESULTS: Phenotypic identification and genotyping by PCR using primers for differentiating DNA markers allowed to attribute 14 isolates to B. melitensis biovar 2, and 7 - to B. abortus biovar 3. By using the MLVA method, connection of MLVA genotypes of 9 Brucella isolates with their reservoir hosts (sheep, cows) was shown providing their circulation in Khentii, Bulqan, and Khubsgul provinces bordering with Russia. Nine isolates from different hosts (camel, yaks, goats, sheep) isolated in Ovorkhangai, Dundgovi, and Dornogovi provinces, which have not border with Russia, had closely related MLVA genotypes indicating an opportunity of migration of pathogenic Brucella species to not-typical hosts. CONCLUSION: Molecular-genetic typing of Brucella isolated in Mongolia was done for the first time; levels of their genetic relation and diversity were demonstrated. Circulation of Brucella isolated with specific MLVA genotypes was connected to territories of specific Mongolian provinces. The study proved migration of Brucella to not-typical hosts. Comparative study of isolates circulating in frontier with Mongolia areas of Russia (Irkutsk region, Tyva and Buryat Republics) are necessary to perform.

[Genotyping some Borrelia burgdorferi sensu lato isolates from Ixodes ricinus ticks in Russia and Ukraine].

The 4 Borrelia burgdorferi sensu lato isolates obtained from 1. Ricinus ticks collected in the natural foci in Russia and Ukraine, having an unusual RFLP Msel-pattern, were studied using sequencing rrfA-rrlB spacer and rrs gene. The Ir-5215 isolate from the tick collected in southern Ukraine represented recently described genospecies B. spielmanii pathogenic for humans. The three atypical isolates Ir-3519, Ir-4721, and Ir-4812 had 100% identity with the sequence of the atypical European B. burgdorferi sensu stricto strains. They constituted a subgroup of the B. burgdorferi sensu stricto on the grounds of Multilocus sequence analysis (MLSA). These data can be indicative of the genetic heterogeneity of the current group B. burgdorferi sensu stricto.

Chapter 4. Recent epidemiology of tick-borne encephalitis an effect of climate change?

Consideration is given to the opinion of some specialists that the rise in tick-borne encephalitis (TBE) morbidity at the turn of the century has been accounted for by new features of TBE epidemiology as well as by global climate change. It is shown that neither the reputed current expansion of the ranges of main TBE vectors, the taiga (Ixodes persulcatus) and sheep (Ixodes ricinus) ticks, nor the significant rise of their abundance and TBE virus prevalence in them are confirmed by any objective data. The concept of recent tick expansion to large cities and human TBE infection in newly formed urban foci disagrees with the facts repeatedly described during the past four decades. There is no reliable information on the expansion of TBE nosological range. The influence of newly formed anthropurgic foci and of changes in the contribution of city dwellers to the general morbidity structure on the current epidemiological situation is estimated. As in the case of any other zoonosis with natural focality, the level of epidemiological manifestation of TBE foci is determined by two main parameters: the intensity of virus circulation in the foci (i.e., their loimopotential) and the frequency of human contact with them. Attention is paid to the character of interaction between these two factors, which accounted for a major outbreak of TBE morbidity at the end of the twentieth century, followed by a long-term decrease in its level.

[Diagnostic efficacy of phosphorescent immunochips for serologic diagnostics of tick-borne encephalitis].

AIM: To assess sensitivity and specificity of phosphorescent immunochips developed by the authors on the basis of microplate phosphorescent assay (PHOSPHAN) for detection of IgM and IgG antibodies to tick-borne encephalitis virus (TBEV) in sera of patients and to compare results of PHOSPHAN assay with results obtained by lanthanide immunofluorescence assay (LIFA) and solid-phase enzyme immunoassay (SPEIA). MATERIALS AND METHODS: Two hundred sixty one serum samples were tested, including 155 samples from 74 patients with clinical diagnosis of TBE confirmed by serologic identification of IgM antibodies to TBEV. Sera were collected in 2003 in Perm region from persons, which fell ill during seasonal increased activity of ticks-vectors of TBEV, as well as from healthy blood donors. Phosphorescent immunochip corresponds 96-well plate with 4 active microzones formed on the bottom of each well, which are able to detect specific IgM and IgG antibodies to TBEV. Immune reaction was visualized by conjugate of streptavidin with Pt-coproporphyrin. Intensity of fluorescence was measured by scanning the bottom of previously dried microwell with scanner IFI-02. RESULTS: Comparable sensitivity and specificity of POSHPHAN assay, LIFA and SPEIA was demonstrated for detection of IgM and IgG antibodies to TBEV in samples. Immunoluminescence-based PHOSPHAN assay and LIFA were more sensitive for analysis of sera with low titer of specific IgM antibodies. CONCLUSION: PHOSPHAN assay could be used for early serologic diagnostics of TBE as well as for assessment of antibody level for control of efficacy of treatment in patients with prolonged illness or level of protective immunity in vaccinees.

[Isolation of tick-borne borreliosis agent from blood of patients].

During spring-autumn period of 2006 Borrelia were isolated for the first time in Russia from blood of 79 patients treated in Perm City Clinical Hospital for Infectious Diseases No. 1 with diagnosis "tickborne borreliosis, manifestive form with migrating erythema, localized stage". Ten primary isolates (12.7% of total seeded samples) were obtained by seeding plasma samples on the BSK medium. Their subsequent identification by polymerase chain reaction-restriction fragments length polymorphism revealed presence of Borrelia garinii NT29 in all patients. Length of sequenced fragment of rrfA-rrlB region was 253 b.p. Seven isolates had 100% and 3 - 99.6% similarity with typical strain NT29 (L30130). Nucleotide sequences of 4 obtained isolates were deposited in GenBank database (No. AM932199 - AM932202). It was proposed that B. garinii NT29 more frequently than other Borrelia species can be an etiologic agent of tick-borne borreliosis not only in Perm region but also in whole Russia.

[Microorganisms of the order Rickettsiales in taiga tick (Ixodes persulcatus Sch.) from the Pre-Ural region].

The PCR and sequence analysis revealed DNA Ehrlichia muris, Anaplasma phagocytophilum, and Rickettsia spp. in the I. persulcatus ticks and blood samples from a patients with acute febrile illness occurring after a tick bite, registered in the seasonal peak of the tick activity of one of the highly endemic areas of Russia (Perm region). These data confirmed the validity a diagnosis of HME and HGA, which were made earlier on the basis of the clinical-serologic survey. In 10.0% of the tested taiga ticks were detected DNA of two and more agents in various combinations i.e. E. muris and Rickettsia spp, A. phagocytophilum and Rickettsia spp., and E. muris, A. phagocytophilum and Rickettsia spp. DNA of a R. helvetica was detected in I. persulcatus tick and blood tick-bitten patient with febrile episodes. Probably that R. helvetica can be etiological agent in some part of cases with the serologically unconfirmed diagnoses of acute feverish diseases developing after tick bite.

[Current characteristics of natural nidality of tick-borne encephalitis: new or well forgotten?].

A number specialists' opinion as to which the rise in the incidence of tick-borne encephalitis at the turn of the centuries is due to the new features of the epidemiology of this infection and to global climatic changes is analyzed. There are no objective evidence suggesting the ongoing expansion of a natural habitat of the major vectors--taiga (Ixodes persulcatus) and wood (I. ricinis) ticks and the noticeable increase in their size and virus infection rates. The notion of the recent penetration of ticks into the metropolises where natural focuses have emerged and human beings are infected is inconsistent with the multidescribed facts. There is no significant evidence for the expansion of a nosoarea of tickborne encephalitis. The impact of reformed anthropurgic foci and that of the proportion of town-dwellers in the general structure of morbidity on the epidemic situation have been evaluated. The intensity of an epidemic manifestation of natural foci is always determined by two most important parameters: 1) the loimopotential of foci and 2) the intensity of the population's contact with them. The nature of an interaction between these factors, which has caused a rapid surge of morbidity rates and their subsequent long-time reduction, is considered.

[Difference in amino acid sequences of B. afzelii P66 surface-exposed loop region].

In the present work, we performed a phenotyping analysis of 45 B. afzelii 89-a.a. long amino acid sequences of 7 different allele variants, corresponding to the surface-exposed loop region of P66. 45 investigated isolates showed 5 phenotypically different variants; 2 phenotypically different variants of loop region, in particular, also showed mutations in the putative monoclonal antibody H1337 binding site; the similarity between the amino acid sequences taken from different variants is about 96.66% to 98.88%; in one natural locus up to 3 different phenotypes of P66 could circulate simultaneously.

[Genetic heterogeneity of Borrelia afzelii in the natural focus of the Middle Urals].

As shown by sequencing the spacer rrf (5S)--rrl (23S) in 72 isolates of B. afzelii (one of the causative agents of Ixodes tick borne Borrelia infections) and the chromosomal gene coding protein P66 in 22 isolates, that in the natural focus located in the Middle Urals two different genetic subgroups (VS461 and NT28) of this genospecies simultaneously circulate. These subgroups are represented by 5 gene variants (rrf) 5S--(rrl) 23S and 5 allelic variants in gene p66. The latter, similarly to spacer gene variants, are not linked with a definite host and occur in different rrf--rrl variants of the infective agent. At the same time the definite species of vectors and carriers may be the host of several different B. afzelii variants, both in the spacer and in the gene coding protein P66, which maintains the genetic heterogeneity of B. afzelii population in the natural focus.

[Allele variants of Borrelia afzelii found by sequencing of the chromosome gene p66].

Sequencing of gene p66 fragments of 246-337 p.b. was performed in 45 isolates of Aorrelia afzelii isolated from different transmitting agents and reservoir hosts within the habitation area of the spirochaeta. At least seven allele variants of the pathogenic agent were found indifferent natural foci of the disease. The extent of similarity between the nucleotide sequences of the isolates of the same allele variant was 99.9-100%; the extent of similarity between different allele variants was 98.9-99.7%. It was found that the majority of genovariants of A. afzelii (with respect to 5S-23S) incorporated several different allele variants of gene p66, all allele variants being found in the two genogroups (VS461 and NT28) of the pathogenic agent.