Genetic variability of Rickettsia spp. in Ixodes persulcatus/Ixodes trianguliceps sympatric areas from Western Siberia, Russia: Identification of a new Candidatus Rickettsia species.

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.

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.

Genetic variability of Anaplasma phagocytophilum in ticks and voles from Ixodes persulcatus/Ixodes trianguliceps sympatric areas from Western Siberia, Russia.

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.

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.

Genetic diversity of Anaplasma and Ehrlichia in the Asian part of Russia.

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.

Tickborne pathogen detection, Western Siberia, Russia.

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.