Natalya N. Livanova

Tickborne Pathogen Detection, Western Siberia, Russia

Ixodes and Dermacentor ticks harbor Borrelia, Anaplasma/Ehrlichia, Bartonella, and Babesia species.

Genetic Variability of Anaplasma phagocytophilum in Ixodes persulcatus Ticks and Small Mammals in the Asian Part of Russia

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.

Comparative Metagenomic Profiling of Symbiotic Bacterial Communities Associated with Ixodes persulcatus, Ixodes pavlovskyi and Dermacentor reticulatus Ticks

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.

The study of heterogeneity of 16S rRNA gene and groESL operone in DNA samples of Anaplasma phagocytophilum, Ehrlichia muris, and “Candidatus Neoehrlichia mikurensis” determined in Ixodes persulcatus ticks on the territory of Ural, 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-HGE1, 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 of A. 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).

Distribution of the Ticks Ixodes persulcatus and Ixodes pavlovskyi on the Boundary of the Forest and Forest-Steppe Zones in the Ob Region

Surveys of ixodoid ticks were performed in Novosibirsk Province (Novosibirsk and Toguchin Districts) and in the vicinity of Akademgorodok (Novosibirsk) in 2009–2010. The abundance and distribution of ticks were assessed in 8 types of habitats. Ixodes persulcatus (Schulze, 1930) was collected by flagging in Novosibirsk and Toguchin Districts, with the highest densities of 19 ind./km being observed in habitats with small-leaved trees. Three species of ticks: Ixodes persulcatus, I. pavlovskyi (subspecies I. pavlovskyi occidentalis Filip. et Pan., 1998), and Dermacentor reticulatus (Fabricius, 1794) were recorded in a recreational forest of Akademgorodok. A high abundance (22 ind./km) of I. pavlovskyi was observed in pine forests subjected to considerable recreational load. The abundance of I. persulcatus was the highest in aspen-birch and birch-aspen forests. D. reticulatus was captured in pine forests and fallow lands, its abundance varying from 0.2 to 2 ind./km.

Age-related changes in natural populations of Ixodes pavlovskyi (Ixodinae) from Novosibirsk and its environs

The age structure of I. pavlovskyi natural populations from Novosibirsk and its environs was investigated on the basis of fat reserves in the midgut and the fat body of the ticks.

Detection of Babesia DNA in small mammals and ixodid ticks in the North Urals, Western Siberia, and Far East of Russia

A total 932 small mammals and 458 questing adult Ixodes persulcatus from Sverdlovsk, Novosibirsk regions and Khabarovsk Territory as well as 128 Haemaphysalis japonica, 34 H. concinna and 29 Dermacentor silvarum from Khabarovsk Territory was examined on the Babesia presence by nested PCR based on the 18S rRNA gene. Babesia microti DNA were found in samples from small mammals in all studied regions — in 36.2% of samples from Sverdlovsk region, in 5.3% of samples from Novosibirsk region and in 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 the sequence of B. microti strain Munich. B. microti were found most frequently in samples from Myodes spp., they were found also in Microtus spp., Apodemus spp., Sorex spp. and Sicista betulina. One from 347 analyzed I. persulcatus from Novosibirsk region and one from 77 I. persulcatus from Khabarovsk Territory were shown to contain B. microti US-type DNA. One I. persulcatus from Novosibirsk region have contained B. divergens DNA. This is the first determination of B. divergens in I. persulcatus and the first determination of B. microti in I. persulcatus in Asian part of Russia. Three novel genetic variants of Babesia sensu stricto were revealed in three H. japonica from Khabarovsk Territory. One novel Babesia 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 Babesia crassa (97.1–97.6% similarity); they clustered together with ruminant pathogens B. crassa and Babesia major.