Search

Objectives: Studies report that women with opioid use disorder experience more comorbid psychiatric disorders and behavioral health burdens than men. We broadened the study of this phenomenon to alcohol use disorder (AUD) and other substance use disorders (SUDs) using national epidemiologic data., Methods: Data from the National Epidemiological Survey on Alcohol and Related Conditions Wave-III identified 2491 women and 3317 men with DSM-5 AUD or SUDs, and 30,501 men and women without such disorders. Women and men with AUD and SUDs were compared to those without such disorders on 22 measures of DSM-5 psychiatric disorders and behavioral histories (eg, incarceration and suicide attempt). The interaction of female sex and AUD in relation to these measures was evaluated and we repeated analyses for SUDs., Results: Separate analyses of female and male adults with and without AUD or SUDs showed AUD and SUDs in both sexes were strongly associated with psychiatric disorders and behavioral histories. In pooled analyses, the interaction of female sex and AUD showed women with AUD had significantly greater likelihood than men of: borderline personality disorder, pain; parental history of incarceration; traumatic events; incarceration; and lifetime homelessness (P < 0.05 for all), but not for psychiatric diagnoses. Interaction of female sex with SUDs showed women had increased likelihood of all personality disorders, traumatic events, and past incarceration ( P < 0.05 for all)., Conclusions: Certain personality disorders and behavioral histories, but not psychiatric diagnoses, may play a distinct role in leading to SUDs among women when compared to men., Competing Interests: The authors report no conflicts of interest., (Copyright © 2021 American Society of Addiction Medicine.)

Background: The associations of current and remitted cannabis use disorder (CUD) with health-related quality of life (HRQOL) and employment have not been studied, and we aim to address these gaps., Methods: The 2012-2013 National Epidemiological Survey on Alcohol and Related Conditions III (NESARC III) surveyed a nationally representative sample of non-institutionalized civilian US adults (≥18 years) (n = 36,309 unweighted). Using DSM-5 criteria, adults with current CUD were compared with those with CUD in remission and no history of CUD on standard measures of the mental and physical component scores of HRQOL and of quality-adjusted life years (QALYs) along with employment in the past 12 months. Multivariable-adjusted regression analyses were used to adjust for and examine the role of covariates., Results: Overall, 2.5% of the study sample, representing 6.0 million adults nationwide, met criteria for current CUD, and 3.7%, representing 8.8 million adults, met the criteria for CUD in remission. Adults with current or past CUD had lower mental HRQOL and QALYs, as compared to adults who never had CUD. However, these differences were no longer significant when adjusted for behavioral co-morbidities and personal histories. Current CUD was associated with lower odds of being employed (Adjusted odds ratio AOR = 0.76; 95% confidence intervals [CI], 0.60-0.96), but CUD in remission with a greater likelihood of employment (Adjusted odds ratio = 1.53; 95% CI, 1.23-1.91), both as compared to those never experiencing CUD., Conclusions: Both current CUD and past CUD are adversely associated with HRQOL and current CUD with not being employed; Since CUD associations are not independent of comorbidities, treatment must take a wide-ranging approach., Competing Interests: Each author completed and submitted the ICMJE form for disclosure of potential conflicts of interest., (Copyright © 2021 American Society of Addiction Medicine.)

Rickettsia spp. are the causative agents of a number of diseases in humans. These bacteria are transmitted by arthropods, including ixodid ticks. DNA of several Rickettsia spp. was identified in Ixodes persulcatus ticks, however, the association of Ixodes trianguliceps ticks with Rickettsia spp. is unknown. In our study, blood samples of small mammals (n=108), unfed adult I. persulcatus ticks (n=136), and I. persulcatus (n=12) and I. trianguliceps (n=34) ticks feeding on voles were collected in two I. persulcatus/I. trianguliceps sympatric areas in Western Siberia. Using nested PCR, ticks and blood samples were studied for the presence of Rickettsia spp. Three distinct Rickettsia species were found in ticks, but no Rickettsia species were found in the blood of examined voles. Candidatus Rickettsia tarasevichiae DNA was detected in 89.7% of unfed I. persulcatus, 91.7% of engorged I. persulcatus and 14.7% of I. trianguliceps ticks. Rickettsia helvetica DNA was detected in 5.9% of I. trianguliceps ticks. In addition, a new Rickettsia genetic variant was found in 32.4% of I. trianguliceps ticks. Sequence analysis of the 16S rRNA, gltA, ompA, оmpB and sca4 genes was performed and, in accordance with genetic criteria, a new Rickettsia genetic variant was classified as a new Candidatus Rickettsia species. We propose to name this species Candidatus Rickettsia uralica, according to the territory where this species was initially identified. Candidatus Rickettsia uralica was found to belong to the spotted fever group. The data obtained in this study leads us to propose that Candidatus Rickettsia uralica is associated with I. trianguliceps ticks., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Ixodes persulcatus, Ixodes pavlovskyi, and Dermacentor reticulatus ticks inhabiting Western Siberia are responsible for the transmission of a number of etiological agents that cause human and animal tick-borne diseases. Because these ticks are abundant in the suburbs of large cities, agricultural areas, and popular tourist sites and frequently attack people and livestock, data regarding the microbiomes of these organisms are required. Using metagenomic 16S profiling, we evaluate bacterial communities associated with I. persulcatus, I. pavlovskyi, and D. reticulatus ticks collected from the Novosibirsk region of Russia. A total of 1214 ticks were used for this study. DNA extracted from the ticks was pooled according to tick species and sex. Sequencing of the V3-V5 domains of 16S rRNA genes was performed using the Illumina Miseq platform. The following bacterial genera were prevalent in the examined communities: Acinetobacter (all three tick species), Rickettsia (I. persulcatus and D. reticulatus) and Francisella (D. reticulatus). B. burgdorferi sensu lato and B. miyamotoi sequences were detected in I. persulcatus and I. pavlovskyi but not in D. reticulatus ticks. The pooled samples of all tick species studied contained bacteria from the Anaplasmataceae family, although their occurrence was low. DNA from A. phagocytophilum and Candidatus Neoehrlichia mikurensis was first observed in I. pavlovskyi ticks. Significant inter-species differences in the number of bacterial taxa as well as intra-species diversity related to tick sex were observed. The bacterial communities associated with the I. pavlovskyi ticks displayed a higher biodiversity compared with those of the I. persulcatus and D. reticulatus ticks. Bacterial community structure was also diverse across the studied tick species, as shown by permutational analysis of variance using the Bray-Curtis dissimilarity metric (p = 0.002). Between-sex variation was confirmed by PERMANOVA testing in I. persulcatus (p = 0.042) and I. pavlovskyi (p = 0.042) ticks. Our study indicated that 16S metagenomic profiling could be used for rapid assessment of the occurrence of medically important bacteria in tick populations inhabiting different natural biotopes and therefore the epidemic danger of studied foci.

Recently, a new Ehrlichia genetic variant, Ehrlichia sp. Khabarovsk, was identified in tissue samples of small mammals captured in the Russian Far East. To further characterize Ehrlichia sp. Khabarovsk, tissue homogenate from a naturally infected gray red-backed vole (Myodes rufocanus) was passaged three times in newborn laboratory mice. Using nested PCR Ehrlichia sp. Khabarovsk DNA was detected in tissue samples from infected mice at 1-4 weeks post inoculation. Electron microscopic examination revealed morulae containing gram-negative bacterial cells in monocytes of mouse spleen and liver. The size and ultrastructure of these cells corresponded to those described previously and allowed us to identify the bacteria as Ehrlichia sp. The comparison of ehrlichial 16S rRNA, groEL and gltA genes and putative GroEL and GltA amino acid sequences has demonstrated that Ehrlichia sp. Khabarovsk, like Ehrlichia ruminantium, is more distant from all other Ehrlichia species than these species are between themselves. Phylogenetic analysis has shown that Ehrlichia sp. Khabarovsk belongs to the clade formed by Ehrlichia spp. but clusters separately from other Ehrlichia species and genetic variants. These data indicate that Ehrlichia sp. Khabarovsk can be considered as a new candidate species. We propose to designate it as 'Candidatus Ehrlichia khabarensis' according to the territory where this species was found., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

To study Babesia diversity in Ixodid ticks in Russia, Ixodes persulcatus, Haemaphysalis japonica, Haemaphysalisconcinna, Dermacentor silvarum, and Dermacentor nuttalli ticks collected in the Far East and Baikal region were assayed for the presence of Babesia spp. using nested PCR. In total, Babesia DNA was detected in 30 of the 1125 (2.7%) I. persulcatus, 17 of the 573 (3.0%) H. concinna, and 12 of the 543 (2.2%) H. japonica but was undetectable in any of the 294 analyzed Dermacentor spp. Partial 18S rRNA gene sequences were determined for all of the positive samples. Among the positive ticks, nine I. persulcatus were infected by Babesia microti 'US'-type, five I. persulcatus were infected by Babesia divergens-like parasites, and 11 I. persulcatus were infected by Babesia venatorum. For all three of these species, the determined 18S rRNA gene sequences were identical to those of the Babesia genetic variants found previously in I. persulcatus in Russia. In addition, five I. persulcatus from the Baikal region and all of the positive Haemaphysalis spp. ticks carried 13 different sequence variants of Babesia sensu stricto belonging to distinct phylogenetic clusters. Babesia spp. from 29 ticks of different species collected in distinct locations belonged to the cluster of cattle and ovine parasites (Babesia crassa, Babesiamajor, Babesiamotasi, Babesiabigemina, etc.). Babesia spp. from four H. japonica ticks in the Far East belonged to the cluster formed by parasites of carnivores. One more Babesia sequence variant detected in an I. persulcatus tick from the Baikal region belonged to the cluster formed by parasites of cattle and wild cervids (B. divergens, Babesiacapreoli, B. venatorum, Babesiaodocoilei, etc.)., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Anaplasma phagocytophilum is a causative agent of granulocytic anaplasmosis in different mammals. The presence of A. phagocytophilum was assayed in Ixodes persulcatus, Ixodes trianguliceps ticks and Myodes spp. voles from two I. persulcatus/I. trianguliceps sympatric areas in the Omsk region (Western Siberia, Russia). In total, A. phagocytophilum was found in 42/108 (38.9%) of vole blood samples, 13/34 (38.2%) of I. trianguliceps ticks removed from voles, 1/12 (8.3%) of I. persulcatus removed from voles, and 18/279 (7.2%) of questing I. persulcatus. GroESL operon sequence analysis of positive samples revealed three distinct A. phagocytophilum genetic groups previously identified in ticks and mammals in Russia. Genetic group 1 was found in 6/36 (16.7%) of sequenced positive blood samples; this group was previously revealed in I. persulcatus and Myodes spp. voles in different regions of Russia. Genetic group 2 was found in 30/36 (83.3%) of sequenced positive blood samples and all positive I. trianguliceps; this group was previously revealed only in Myodes spp. voles and common shrews (Sorex araneus) in I. persulcatus/I. trianguliceps sympatric areas in the Northern Ural. Genetic group 3 was found in all positive questing I. persulcatus and one blood sample; this group was previously revealed in I. persulcatus and Siberian chipmunks (Tamias sibiricus). We suppose that I. trianguliceps is the most probable vector for A. phagocytophilum of group 2. Analysis of the msp4 gene, intergenic region DOV1, and some other genetic loci has shown that isolates from different genetic groups significantly differ in all studied loci and that A. phagocytophilum of group 2 is closely related to A. phagocytophilum isolates revealed in voles and I. trianguliceps in Europe. A. phagocytophilum of groups 1 and 2 are the most similar to each other, while A. phagocytophilum of group 3 clusters with European A. phagocytophilum isolates from I. ricinus and various mammalian species., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Microtine rodents were captured in two disconnected sampling sites in Omsk region where Ixodes pesulrcatus and Ixodes trianguliceps are sympatric. In blood samples of rodents the DNA was revealed belonging to several ixodid-transmitted pathogens: Borrelia burgdorferi sensu lato (prevalence 20.0 and 6.0%, here and further values are given for the first and second site, respectively), Borrelia miyamotoi (8.3 and 2.0%), Anlaplasnma phagocytophilum (33.3 and 48.0%), Ehrlichia muris (30.0 and 2.0%) and Babesia microti (33.3 and 42.0%). Three genetic groups of A. phagocytophilhm based on 16S rRNA gene and groESL operon, as well as two genetic groups of B. microti, B. microti 'US'-type and B. microti 'Munich'-type, were detected.

Totally 484 Haemaphysalis japonica, 359 Haemaphysalis concinna and 221 Dermacentor silvarumn collected in Amur region and Khabarovsk Territory of the Russian Far East were examined on the presence of Anaplasmataceae bacteria using nested PCR. All positive samples were characterized by analysis of the 16S rRNA gene and/or groESL operone nucleotide sequences. Forty nine H. japonica and three H. concinna were shown to contain DNA of two new Ehrlichia genetic variants. These genetic variants on the basis of the 16S rRNA gene and groESL operone nucleotide sequences analysis were most closely related to Ehrlichia spp. revealed in Haemaphysalis spp. ticks in Japan. Four H. concinna from Amur region were shown to contain DNA of a new Anaplasma bovis genetic variant, which corresponded to A. bovis genetic variant revealed in a red gray-backed vole and a Siberian chipmunk from the Far East. Three H. concinna and nine D. silvarum contained DNA of non-typical bacteria which can't be attributed to any Anaplasmataceae genera based on the determined sequences of the 16S rRNA gene fragments. The revealed non-typical bacteria on the basis of 16S rRNA gene sequences significantly differed from each other and didn't form a separate genetic group.

The analysis was applied to detect DNA of agents of human granulocytic anaplasmosis and monocytic erlychiosis. The sampling included 109 ticks of Ixodes species from Novosibirsk oblast and Khabarovsk kray and blood samples of 111 mouse-like rodents from Omsk oblast. The used techniques included polymerase chain reaction in real-time operation mode with set of reagents "RealBest DNA Anaplasma phagocytophilum/Ehrlichia muris, ehrlichia chaffeensis" ("Vector-Best" Novosibirsk) and double round polymerase chain reaction. The DNA of A. phagocytophilum, agent of granulocytic anaplasmosis and/or DNA of E. muris, agent of monocytic erlychiosis was detected in 21 ticks and in blood samples of 52 voles. Both techniques were applied. The DNA of A. phagocytophilum was detected in samples of 2 voles and in 1 tick only after polymerase chain reaction in real-time operation mode was applied. It demonstrated that the set of reagents "RealBest DNA Anaplasma phagocytophilum/Ehrlichia muris, ehrlichia chaffeensis" permits to detect the DNA of isolates of A. phagocytophilum subsumed to different genetic groups. The set can be used for fast and effective detection of the DNA of agents of agents of human granulocytic anaplasmosis and monocytic erlychiosis in suspensions of analyzed ticks and blood samples.

The specimens of 3552 questing adult Ixodes persulcatus and 1698 blood/tissue samples of small mammals collected in Ural, Siberia, and Far East of Russia were assayed for the presence of Anaplasma phagocytophilum by nested PCR based on the 16S rRNA gene. Totally, A. phagocytophilum was detected in 112 tick and 88 mammalian samples. The nucleotide sequences of the 16S rRNA gene and groESL operon (1244-1295 bp) were determined for A. phagocytophilum samples from 65 ticks and 25 small mammals. Six different 16S rRNA gene variants differing by 1-5 nucleotide substitutions were detected, and only one variant matched the sequences deposited in GenBank. Analysis of groESL sequences allowed the A. phagocytophilum samples to be divided into three groups; moreover, the samples from different groups also differed in the 16S rRNA gene sequences. The A. phagocytophilum sequences from group I were detected in 11 Myodes spp. samples from West Siberia and Far East and in 19 I. persulcatus samples from all examined regions; from group II, in 10 samples of Myodes spp. and common shrews (Sorex araneus) from Ural; and from group III, in four samples of Asian chipmunks (Tamias sibiricus) from West Siberia and Far East; and in 46 I. persulcatus samples from all examined regions. The nucleotide sequences of A. phagocytophilum groESL operon from groups I and II were strictly conserved and formed with A. phagocytophilum groESL sequence from a Swiss bank vole (Myodes glareolus) (GenBank accession no. AF192796), a separate cluster on the phylogenetic tree with a strong bootstrap support. The A. phagocytophilum groESL operon sequences from group III differed from one another by 1-4 nucleotides and formed a separate branch in the cluster generated by European A. phagocytophilum strains from roe deer (Capreolus capreolus) and Ixodes ricinus ticks.

Objective: The aim of this work was to study the prevalence and genetic diversity of Babesia in Ixodes persulcatus ticks and small mammals from Ural and Siberia in Russia., Methods: In total, 481 small mammals and 922 questing adult I. persulcatus from North Ural (Sverdlovsk region) and West Siberia (Novosibirsk region) were examined for the presence of Babesia by nested PCR based on the 18S rRNA gene., Results: Babesia microti of the 'Munich'-type was found in 36.2% of blood samples of the small mammals from the Sverdlovsk region and B. microti of the 'US'-type in 5.3% of the animals from the Novosibirsk region. Babesia DNA was not detected in 133 analysed I. persulcatus from the Sverdlovsk region; however, it was found in 24 of 789 ticks from the Novosibirsk region. Three distinct Babesia species were detected in I. persulcatus. B. microti 'US'-type was identified in 10 ticks, Babesia closely related to B. divergens/B. capreoli in 2 ticks, and Babesia closely related to B. venatorum (EU1) in 12 ticks., Conclusion: To our knowledge, this is the first detection of Babesia sensu stricto in I. persulcatus ticks and of B. microti in I. persulcatus in the Asian part of Russia.

A total of 3552 Ixodes persulcatus from Sverdlovsk, Chelyabinsk, Novosibirsk, Irkutsk regions and Khabarovsk Territory were examined on the Ehrlichia and Anaplasma presence by nested PCR based on the 16S rRNA gene. Both Anaplasma phagocytophilum and Ehrlichia muris DNA were found in I. persulcatus in all studied regions. A. Phagocytophilum was detected in 1.3-6.3% of ticks and E. muris - in 2.0-14.1% of ticks. Moreover, "Candidatus Neoehrlichia mikurensis" DNA was found in 8 ticks collected in Novosibirsk, Irkutsk Regions and Khabarovsk Territory. Partial nucleotide sequences of 16S rRNA gene and groESL operone (1240-1300 bp) were determined for 65 samples of A. Phagocytophilum, 17 samples of E. muris and 4 samples of "Candidatus Neoehrlichia mikurensis". Nucleotide sequences of 16S rRNA gene and groESL operone of E. muris and "Candidatus Neoehrlichia mikurensis" were shown to be highly conservative, and nucleotide sequences of groESL operone of both E. muris and "Candidatus Neoehrlichia mikurensis" differed from the sequences found previously in other species of Ixodid tick. On the basis of analysis of the 16S rRNA gene and groESL operone sequences it was concluded that all revealed samples A. Phagocytophilum could be divided into 2 groups. GroESL operone sequences of A. Phagocytophilum from the first group were identical to each other but significantly differed from the known groESL operone sequences (less than 98.2% of similarity), whereas their 16S rRNA gene sequences were identical to the sequence of widely distributed and pathogenic for human A. Phagocytophilum genetic variant (CAHU-HGEl, GenBank AF093788) or differed from it by a single nucleotide substitution. The nucleotide sequences of groESL operone of A. Phagocytophilum from the second group differed from each other by 1-4 nucleotides and were closely related (99.2-99.4% of similarity) to the sequences of groESL operone ofA. phagocytophilum isolates found in Europe in Ixodes ricinus and roe deer. The nucleotide sequences of the 16S rRNA gene of A. Phagocytophilum from the second group were most similar to the sequence of the rare A. Phagocytophilum genetic variant previously found only in China (GenBank DQ342324).

There has been recently a rise in referrals for Ixodes tick bites in the spring and summer periods in the Kamchatka Territory. Among the dominant tick species, there has been the taiga tick Ixodes persulcatus habiting the extensive areas of the southern and central parts of the peninsula. Examination of 84 I. persulcatus females collected from human beings and domestic animals in 2003 to 2007 detected DNA of the pathogens of tick-borne borreliosis (B. burgdorferi sensu lato), rickettsiasis (R. tarasevichiae and R. helvetica), and Ehrlichiosis/anaplasmosis (A. phagocytophilum). Tick-borne encephalitis RNA and antigens and babesiasis DNA were not found in the study samples. Despite the small number of taiga ticks in Kamchatka, the detection of the pathogens of various infectious diseases in the ticks suggests that there may be a risk for contamination of the peninsula's population with these pathogens.

Totally, 2590 questing adult Ixodes persulcatus ticks and 1458 small mammals from Ural, Siberia, and the Far East as well as 53 Haemaphysalis concinna, 136 Haem. japonica, and 43 Dermacentor silvarum ticks--exclusively adults--from the Far East were examined for the presence of Ehrlichia and Anaplasma by nested PCR based on the 16S rRNA gene. Both Anaplasma phagocytophilum and Ehrlichia muris were found in I. persulcatus and small mammals from all the studied regions. Myodes spp., Microtus spp., Sorex araneus, Apodemus peninsulae, and Tamias sibiricus were naturally infected with An. phagocytophilum and E. muris. Five of the examined I. persulcatus and 5 of the examined wild rodents from Siberia and the Far East were infected with 'Candidatus Neoehrlichia mikurensis'. The determined 16S rRNA gene sequences of 'Candidatus Neoehrlichia mikurensis' were identical to the sequences of Japanese isolates, while the determined groESL sequences were unique. A new Ehrlichia sp. variant closely related to the Ehrlichia sp. EHf669 found in Haem. flava from Japan was detected in 11% of Haem. japonica ticks. New Anaplasmataceae bacteria genetically distinct from the known species of this family were found in 3 adult Derm. silvarum from the Far East and in 2 I. persulcatus from Siberia and the Far East. In the Far East, about 15% of the captured small mammals were naturally infected with recently discovered Ehrlichia sp. Khabarovsk. Ehrlichia sp. Khabarovsk was found in about 20% of Myodes spp. and S. araneus but was undetectable in any of the 236 studied Ap. peninsulae. A three-year study has demonstrated that An. phagocytophilum and E. muris were detectable in small mammals from the Far East captured only after the beginning of the tick activity season, from May to November. Ehrlichia sp. Khabarovsk was found in mammals trapped in all the examined periods, from February to November., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Totally, 932 small mammals and 458 questing adult Ixodes persulcatus from Sverdlovsk and Novosibirsk regions and Khabarovsk Territory, as well as 128 Haemaphysalis japonica, 34 H. concinna and 29 Dermacentor silvarum from Khabarovsk Territory were examined for the presence of Babesia by nested PCR based on the 18S rRNA gene. Babesia microti DNA was found in samples of small mammals from all the studied regions--in 36.2% of samples from Sverdlovsk region, 5.3% of samples from Novosibirsk region, and 6.7% of samples from Khabarovsk Territory. The determined B. microti 18S rRNA gene sequences from Novosibirsk region (6 sequences) and from Khabarovsk Territory (10 sequences) were identical to each other and to the sequences of pathogenic for human B. microti US-type, while the determined B. microti 18S rRNA gene sequences from Sverdlovsk region (12 sequences) were identical to those of B. microti strain Munich. B. microti were found most frequently in samples of Myodes spp., they were found also in Microtus spp., Apodemus spp., Sorer spp., and Sicista betulinav. It was shown that one of 347 analyzed I. persulcatus from Novosibirsk region and one of 77 I. persulcatus from Khabarovsk Territory contained B. microti US-type DNA. One I. persulcatus from Novosibirsk region contained B. divergens DNA. In this work B. divergens was for the first time determined in I. persulcatus and B. microti in I. persulcatus in Asian part of Russia. Three different genetic variants of Babesia sensu stricto were found in three H. japonica from Khabarovsk Territory. The first genetic variant was closely related to Babesia sp. revealed in a feral raccoon in Japan (99.9% similarity on the basis of 18S rRNA gene sequences). Two others Babesia genetic variants were most similar to the ovine pathogen Babesia crassa (97.1-97.6% similarity on the basis of 18S rRNA gene sequences).

To determine DNA of herpes simplex virus (HSV), types 1 and 2, the polymerase chain reaction (PCR) method was developed with the subsequent detection of amplification products by means of electrophoresis or the molecular hybridization of nucleic acids (MHNA). Two variants of MHNA have been compared: hybridization in the solution of a biotinylated probe with digoxigenin-labeled PCR with the subsequent sorption of hybridization complexes onto streptavidin-covered plates and solid-phase hybridization of digoxigenin-labeled PCR with a biotinylated probe. Effective hybridization was observed after the denaturation of targets at 95 degrees C in the solution of 50 mM NaOH, but not in neutral solutions. To increase the level of sensitivity of hybridization in solution, the exact selection of the amount of the probe was shown to be necessary, for both its excess and deficiency essentially decreased the method sensitivity. A decrease in the ionic power of hybridization solutions from 6 h SSC to 1 h SSC led to greater specificity of hybridization without a decrease in the method sensitivity. A rise in the temperature of hybridization and subsequent washing to 45 degrees C decreased the sensitivity of the method. The limit of the sensitivity PCR with electrophoretic detection was 30 HSV genome equivalents, and 10 genome equivalents in the presence MHNA in the solution and on the solid phase.

Ixodes persulcatus (n = 125) and Dermacentor reticulatus (n = 84) ticks from Western Siberia, Russia, were tested for infection with Borrelia, Anaplasma/Ehrlichia, Bartonella, and Babesia spp. by using nested polymerase chain reaction assays with subsequent sequencing. I. persulcatus ticks were infected with Borrelia burgdorferi sensu lato (37.6% +/- 4.3% [standard deviation]), Anaplasma phagocytophilum (2.4% +/- 1.4%), Ehrlichia muris (8.8% +/- 2.5%), and Bartonella spp. (37.6% +/- 4.3%). D. reticulatus ticks contained DNA of B. burgdorferi sensu lato (3.6% +/- 2.0%), Bartonella spp. (21.4% +/- 4.5%), and Babesia canis canis (3.6% +/- 2.0%). Borrelia garinii, Borrelia afzelii, and their mixed infections were observed among I. persulcatus, whereas B. garinii NT29 DNA was seen in samples from D. reticulatus. Among the I. persulcatus ticks studied, no Babesia spp. were observed, whereas B. canis canis was the single subspecies found in D. reticulatus.

Babesia infection was studied in 21 blood samples of dogs with symptoms of babesiosis and among 72 Dermacentor reticulatus and 70 Ixodes persulcatus ticks from southwestern Siberia, Russia. Babesia DNA was detected by hemi-nested PCR based on the 18S rRNA gene with subsequent direct sequencing. All of the analyzed canine blood samples and three D. reticulatus, but none from I. persulcatus ticks studied were shown to contain Babesia DNA. Nucleotide sequences of the Babesia 18S rRNA gene fragment of 354 bp long for all 24 positive samples appeared to belong to the subspecies Babesia canis canis and differed only at three positions. The Babesia nucleotide sequences from 17 canine blood samples and from one D. reticulatus tick were identical to each other and to previously known B. canis canis from canine blood in Slovenia. Four canine blood samples and the second tick sample contained a mixture of two nucleotide sequences previously found in canine blood. B. canis canis nucleotide sequence from the third tick differed in the unique nucleotide transition and could correspond to a new genetic variant. Thus, the main etiological agent of canine babesiosis in Novosibirsk region is B. canis canis, and D. reticulatus, but not I. persulcatus, ticks could serve as a vector of this infectious agent. To our knowledge, this is the first report of the B. canis canis nucleotide sequences from ticks.

To evaluate the level of U. urealyticum colonization of female urogenital tract, the method of the multiplex polymerase chain reaction (PCR) in the presence of two pairs of primers, corresponding to genes controlling U. urealyticum 16S rRNA and unique human osteopontin was used. The study of 93 clinical specimens showed no correlation between high colonization level and the presence of definite clinical manifestations of U. urealyticum infection. The determination of ureaplasmic biovars was carried out by the method of PCR in the presence of 3 primers corresponding to the multiple-banded antigen (MBA) gene. Biovar parvo was detected in 85% of the specimens, biovar T960 in 11% and both biovars were detected in 4% of the specimens. The biovar distribution in the groups of women with different clinical symptoms was approximately similar. U. urealyticum of biovar T960 occurred more frequently (33% of the specimens) only in a group of women with vaginal discharge characteristic of inflammation.

Interaction with eukaryotic TATA-binding protein (TBP) was analyzed for natural Escherichia coli RNA polymerase or the recombinant holoenzyme, minimal enzyme, or its sigma subunit. Upon preincubation of full-sized RNA polymerase with TBP and further incubation with a constant amount of 32P-labeled phosphamide derivative of a TATA-containing oligodeoxyribonucleotide, the yield of the holoenzyme-oligonucleotide covalent complex decreased with increasing TBP concentration. This was considered as indirect evidence for complexing of RNA polymerase with TBP. In gel retardation assays, the holoenzyme, but neither minimal enzyme nor the sigma subunit, interacted with TPB, since the labeled probe formed complexes with both proteins in the reaction mixture combining TBP with the minimal enzyme or the sigma subunit. It was assumed that E. coli RNA polymerase is functionally similar to eukaryotic RNA polymerase II, and that the complete ensemble of all subunits is essential for the specific function of the holoenzyme.

Quantities of Ureaplasma spp DNA were determined by the methods of competitive and multiplex polymerase chain reactions (PCR). The results obtained by each of the methods were shown to be compatible. It was established that the number of Ureaplasma spp. cells per one epithelial cell in the female cervical canal varied from 0.05 to 10.

Tick-borne encephalitis virus (TBEV) strains were isolated from ticks in Western Siberia for 12 years. Molecular hybridization of the 46 viral RNA with the TBEV cDNA and oligonucleotide probes revealed differences between the Siberian and Far Eastern strains. A comparison of the viral E gene fragment nucleotide sequence showed 89-98% homology between Siberian TBEV strains, whereas their similarity with strains from other populations was less than 83%. However, the viral E and NS1 glycoprotein antigenic structures appeared to be conservative because of the degenerate genetic code. This was shown by enzyme-linked immunosorbent assay with the corresponding monoclonal antibodies (MAb). The single exception was the MAb 17C3 against nonstructural glycoprotein NS1, which could distinguish Siberian from Far Eastern strains. Moreover, the neurovirulence differed between strains from the two natural populations. Lower neuroinvasiveness of the Siberian strains in comparison with Far Eastern Sofyin strain might be caused by both E and NS1 glycoprotein mutations.

Tick-borne encephalitis (TBE) virus strains were isolated in West Siberia in the forest-steppe region near the Ob river in 1981-1992. Hybridization of genome RNA of 46 TBE strains with [32P]cDNA of TBE Sofyin strain revealed essential differences in the genomes of West-Siberian and Far-Eastern Sofyin strains of TBE virus. Nucleotide sequences of 6 TBE strains (1348-1503 n.) have been determined. A 89-98% homology of Siberian TBE strains has been shown, while the similarity of the respective fragment of E gene for West Siberian and Sofyin strains was no more than 81%. No significant changes in E gene of TBE strains have been detected over a 12-year period.

To detect C. trachomatis DNA, the polymerase chain reaction (PCR) with the use of primers corresponding to variable sites of rRNA gene 16S was carried out. As the positive control of the reaction, the amplification fragment of gene 16S of rRNA, cloned in the plasmid vector and having the length of 530 nucleotide pairs (n.p.), was used. On its basis 2 kinds of the internal control of the reaction were obtained with the deletion of 110 n.p. (pMOS-Chl420) and the insertion of 930 n.p. (pMOS-Chl1460) within the cloned amplification fragment. The study revealed that the addition of the DNA of pMOS-Chl420 or pMOS-Chl1460 into the reaction mixture did not affect the sensitivity of PCR (0.02 pg of bacterial DNA in the sample) in the detection of C. trachomatis DNA isolated both from the culture of bacterial cells and from clinical samples. But in some cases of the amplification of the DNA of internal control pMOS-Chl420, but not pMOS-Chl1460, was observed in the presence of DNA obtained from clinical samples. It was supposedly linked with a higher sensitivity of Taq DNA-polymerase to the action of inhibitors in the synthesis of high-molecular DNA fragments. The observed high frequency of the inhibition (17%) of PCR makes it expedient to carry out this reaction with the use of the internal control.

Four clones producing monoclonal antibodies inhibiting enzymatic activity were used to localize the functionally important antigenic determinants of T7 RNA polymerase. All antibodies were shown to bind to C-terminal fragment of the protein (residues 589-883). The competition studies showed the specificity of the three antibodies toward one epitope and the fourth antibody to another one. By means of limited cleavage of the RNA polymerase with cyanogen bromide with subsequent electrophoretic separation and immunoblotting the peptides containing antigenic determinants were localized. These are Met861-Ala883 for antibody 4H8 and Met750-Met832 for antibodies 9B2, 3H11 and 2A2.

Synthetic constructions containing a peptide antigenic determinant (C-terminal peptide 205-213 of the surface VP1 protein of the foot-and-mouth disease virus, O1K strain), glucosaminylmuramayl dipeptide (GMDP), and polyionic synthetic carriers were prepared. The polymerized peptide and peptide-BSA conjugates were synthesized as well. Among the constructions obtained only peptide-BSA conjugate proved to be highly immunogenic. Application of synthetic constructions to design immunogenic complexes is discussed.

Human serum albumin has been chemically modified by two different spin pH-sensitive labels of the imidazoline series containing in their structure alkylating and carboxyl groups, respectively. The ESR spectra of spin-labeled proteins are sensitive to pH of the medium. The pK values of spin-labeled proteins measured by the ESR method are: pKI = 3.2 +/- 0.1; pKII = 4.75 +/- 0.1. The resulting macromolecular spin pH probes may be used for measuring the local values of pH by the ESR technique within the pH range of 1.8-6.2.

In experiments on ovariectomized rats cAMP-dependent phosphorylation of the uterine nuclear tissue proteins under the action of estradiol, histamine and cAMP was studied. It was shown, that phosphorylation of the uterine isolated nuclear proteins under the influence of endogenous proteinkinases increases 6 hours after estradiol, histamine and/or cAMP injection to the animals. Proteinkinase activity of the uterine tissue nuclei are higher, when exogenous histone H1 is used as a substrate. The ability of histone H1, isolated from the uterine tissue 6 hours after estradiol, histamine or cAMP injection, to be phosphorylated in vitro with exogenous cAMP-dependent proteinkinase is lowered, that is likely to be resultant of preceding phosphorylation of these proteins in vivo.