Genetic variability of Babesia parasites in Haemaphysalis spp. and Ixodes persulcatus ticks in the Baikal region and Far East of Russia.

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.).

[DNA detection of pathogens transmitted by Ixodid ticks in blood of small mammals inhabiting the forest biotopes in Middle Irtysh Area (Omsk Region, West Siberia)].

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.

[Genetic variability of anaplasmataceae bacteria determined in Haemaphysalis spp. and Dermacentor sp. ticks in the territory of the Russian Far East].

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 application of polymerase chain reaction in real-time operation mode to detect DNA of agents of human granulocytic anaplasmosis and monocytic erlychiosis].

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.

[Study of the heterogeneity of 16s rRNA gene and groESL operone in the dna samples of Anaplasma phagocytophilum, Ehrlichia muris, and "Candidatus Neoehrlichia mikurensis" determined in the Ixodes persulcatus ticks in the area of Urals, Siberia, and far east 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).

Genetic diversity of Babesia in Ixodes persulcatus and small mammals from North Ural and West Siberia, Russia.

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.

[Detection of Babesia spp. DNA in small mammals and ixodic ticks on the territory of north Ural, west Siberia and far east of Russia].

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).

[Detection of Borrelia DNA in the Borrelia burgdorferi sensu lato complex in the blood of patients with Ixodes tick-borne borrelios].

Borrelia DNA was detected by polymerase chain reaction in the blood of patients who had suffered from tick suction. DNA was revealed in 28.7 of the blood samples from patients with the erythematous form of Ixodes tick-born borrelioses (ITBB) and in 14.3% of those diagnosed as having tick-borne encephalitis. Blood Borrelia DNA was not detected in patients with end-stage and chronic ITBB. Comparing the results of detection of DNA with those Borrelia protein antibodies has shown that the antibody titers detectable in the patients having Borrelia DNA are lower than those in the patients with the same form of ITBB and without DNA. The detection of DNA should be accomplished in the first 4 weeks after tick bite.