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•Rickettsia raoultiiwas found mainly in Dermacentor silvarumticks from the Far East.•Rickettsia heilongjiangensiswas found mainly in Haemaphysalis concinnaticks.•Rickettsia canadensiswas found for the first time in a H. japonica tick.•R. aeschlimanniiwas first found in H. concinna and H. japonica from the Far East.•CandidatusR. principis and CandidatusR. rara were genotyped at several genes.

Correct identification of tick species is an essential requirement for any scientific study engaged in tick-associated research. However, morphological identification can lead to misinterpretations, especially when dealing with vector-host research and sub-adult, engorged or damaged specimens. To overcome this limitation, we developed a novel assay to discriminate between Ixodes ricinus, I. persulcatusand I. triangulicepsspecies collected from rodents or vegetation, using the second internal transcribed spacer (ITS2) as a genetic marker. This single tube multiplex PCR allows specific amplification of targeted species and produces rapid and accurate results. The specificity was confirmed by sequencing the ITS2 and partial 16S rRNA genes from ticks collected from Estonia, Latvia, Sweden and Russia. We tested the assay in a large-scale experiment, and a total of 1284 ticks removed from rodents and shrews were successfully identified at species level.

•“CandidatusRickettsia mendelii” was first found in Ixodes pavlovskyiin Asian region.•“Ca.R. mendelii” was genotyped by five genetic loci, including three new ones.•“Ca.R. mendelii” was characterized by single locus and multilocus sequence analysis.•“Ca.R. mendelii” forms a well-supported cluster on each phylogenetic tree.•“Ca.R. mendelii”, like Rickettsia bellii, is a basal group of Rickettsia.

•New Borreliasp. was found in Ixodesspp. and samples from small mammals.•MLST suggests new Borreliaas a putative species “CandidatusB. sibirica”.•Borrelia bavariensiswas first found in molted I. triangulicepsand I. apronophorus.•The same B.bavariensiswere found in I.persulcatus, I.triangulicepsand I.apronophorus.

Anaplasma phagocytophilumis a causative agent of granulocytic anaplasmosis in different mammals. The presence of A. phagocytophilumwas assayed in Ixodes persulcatus, Ixodes triangulicepsticks and Myodesspp. voles from two I. persulcatus/I. triangulicepssympatric areas in the Omsk region (Western Siberia, Russia). In total, A. phagocytophilumwas found in 42/108 (38.9%) of vole blood samples, 13/34 (38.2%) of I. triangulicepsticks removed from voles, 1/12 (8.3%) of I. persulcatusremoved from voles, and 18/279 (7.2%) of questing I. persulcatus. GroESLoperon sequence analysis of positive samples revealed three distinct A. phagocytophilumgenetic 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. persulcatusand Myodesspp. 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 Myodesspp. voles and common shrews (Sorex araneus) in I. persulcatus/I. triangulicepssympatric areas in the Northern Ural. Genetic group 3 was found in all positive questing I. persulcatusand one blood sample; this group was previously revealed in I. persulcatusand Siberian chipmunks (Tamias sibiricus). We suppose that I. triangulicepsis the most probable vector for A. phagocytophilumof group 2. Analysis of the msp4gene, 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. phagocytophilumof group 2 is closely related to A. phagocytophilumisolates revealed in voles and I. triangulicepsin Europe. A. phagocytophilumof groups 1 and 2 are the most similar to each other, while A. phagocytophilumof group 3 clusters with European A. phagocytophilumisolates from I. ricinusand various mammalian species.

•Five Rickettsiaspp. and seven new genovariants cause rickettsioses in Russian Siberia.•R. aeschlimanniiand R. slovacawere detected for the first time in patients in Russia.•R. sibiricaand R. raoultiiare the main rickettsial agents in Novosibirsk province.•Clinical manifestations of R. raoultiiinfection are mainly non-specific.•Ten genovariants of R. raoultiican cause rickettsiosis in humans.

Totally, 2590 questing adult Ixodes persulcatusticks and 1458 small mammals from Ural, Siberia, and the Far East as well as 53 Haemaphysalis concinna, 136 Haem. japonica, and 43 Dermacentor silvarumticks – exclusively adults – from the Far East were examined for the presence of Ehrlichiaand Anaplasmaby nested PCR based on the 16S rRNA gene. Both Anaplasma phagocytophilumand Ehrlichia muriswere found in I. persulcatusand small mammals from all the studied regions. Myodesspp., Microtusspp., Sorex araneus, Apodemus peninsulae, and Tamias sibiricuswere naturally infected with An. phagocytophilumand E. muris. Five of the examined I. persulcatusand 5 of the examined wild rodents from Siberia and the Far East were infected with ‘CandidatusNeoehrlichia mikurensis’. The determined 16S rRNA gene sequences of ‘CandidatusNeoehrlichia mikurensis’ were identical to the sequences of Japanese isolates, while the determined groESLsequences were unique. A new Ehrlichiasp. variant closely related to the Ehrlichiasp. EHf669 found in Haem. flavafrom Japan was detected in 11% of Haem. japonicaticks. New Anaplasmataceaebacteria genetically distinct from the known species of this family were found in 3 adult Derm. silvarumfrom the Far East and in 2 I. persulcatusfrom Siberia and the Far East. In the Far East, about 15% of the captured small mammals were naturally infected with recently discovered Ehrlichiasp. Khabarovsk. Ehrlichiasp. Khabarovsk was found in about 20% of Myodesspp. and S. araneusbut was undetectable in any of the 236 studied Ap. peninsulae. A three-year study has demonstrated that An. phagocytophilumand E. muriswere detectable in small mammals from the Far East captured only after the beginning of the tick activity season, from May to November. Ehrlichiasp. Khabarovsk was found in mammals trapped in all the examined periods, from February to November.

•Eight genetic loci (16,117 bp) of "CandidatusRickettsia uralica" were sequenced.•“CandidatusR. uralica" and "CandidatusR. thierseensis have 99.98% sequence identity.•"CandidatusR. uralica" and "CandidatusR. thierseensis" are likely the same species.

•High prevalence of Anaplasma phagocytophilumand Ehrlichia murisin rodents was shown.•phagocytophilumand E. murisare mainly transmitted to rodents by different tick species.•Three genetic clusters of A. phagocytophilumwere revealed in rodents and ticks.•A genetic group, which was associated with Ixodes trianguliceps, prevailed in rodents.•New genetic variants of E. murisand Neoehrlichia mikurensiswere found.

Wild animals are reservoir hosts for a number of tick-transmitted agents, and long-term persistence of the agents is a key factor for their effective transmission from animal hosts to ticks. To study the persistence of Anaplasmataceae in rodents, 59 adult Myodesspp. voles (M. rutilus, M. rufocanus, and M. glareolus) were captured in Omsk Province, Asian Russia, freed from all ectoparasites, and kept in individual cages. Their blood samples were regularly analyzed for the presence of DNA of Anaplasma phagocytophilum, Ehrlichia muris, and “CandidatusNeoehrlichia mikurensis”. Anaplasma phagocytophilum, E. muris, and mixed infections were found in 29 (49 %), 17 (29 %), and 14 (24 %) voles, respectively. DNA of “CandidatusN. mikurensis” was not identified. Long-term persistence of A. phagocytophilumand E. murisin naturally infected Myodesspp. was shown for the first time. Thus, 12 animals with A. phagocytophilumwere found infected for more than three months and four of them carried the bacterium throughout 22–55 weeks of observation. Four voles were infected with E. murisfor 26–40 weeks. Fragments of groESLoperon were sequenced for genotyping A. phagocytophilumand E. muris. In voles with A. phagocytophilum, the determined sequences belonged to two phylogenetic clusters. Most sequences were identical or closely related to those found in small mammals and Ixodes triangulicepsticks from Western Siberia and European countries; one sequence was previously identified in rodents and Ixodes persulcatusand Ixodes pavlovskyiticks from Asian Russia. Sequences of groESLfragments from E. muriswere close to those determined previously in small mammals and I. persulcatusticks from Siberia. In addition, a new groESLsequence of E. muriswas identified in one vole and the sequence substantially differed from all known corresponding E. murissequences (≥ 14 mismatches).

Genetic variability of I. apronophorusfrom Western Siberia, Russia was examined using the nuclear internal transcribed spacer 2 (ITS2) and mitochondrial 16S rRNA and cytochrome coxidase subunit 1 (cox1) genes and compared to those of Ixodes persulcatusand Ixodes triangulicepsfrom the same site. The I. apronophorussequences demonstrated the highest nucleotide and haplotype diversity for both mitochondrial genes, whereas I. persulcatuswas more variable in the nuclear ITS2. Phylogenetic analysis of the molecular sequence data showed that I. apronophorusdiffered from other Ixodesspecies, including Romanian I. apronophorus. The level of identity between 16S rRNA gene sequences of Siberian and Romanian I. apronophoruswas only 91%; these sequences did not form a monophyletic group, indicating that I. apronophorusfrom Siberia and Romania could be different tick species. The analysis of morphological features of the Siberian I. apronophorusconfirmed their consistency with those for the previously described I. apronophorusspecies. Based on the 16S rRNA and ITS2 sequences, Siberian I. apronophorusclustered together with Ixodes kazakstaniand Ixodes scapularis, which are the recognized members of the Ixodes ricinus-I. persulcatusspecies complex within the subgenus Ixodes, and can be assigned to this complex.

North Asian tick-typhus (NATT), also known as Siberian tick typhus, is the main tick-borne rickettsiosis in Siberia, Russia. Recently, a fatal infection in a four-year-old girl with typical tick-borne rickettsiosis symptoms (fever, rash, eschar at the site of the tick bite, myalgia) and meningeal syndrome was registered. In order to identify the etiology of this infection, blood and brain samples from the patient were examined for the presence of a wide range of tick-transmitted agents and enteric viruses by polymerase chain reaction with subsequent sequencing. Rickettsia sibiricasubsp. sibiricaand “CandidatusRickettsia tarasevichiae” DNA was identified in both blood and brain samples. Rickettsia sibiricawas characterized by the gltA, ompAand ompBgenes, and “CandidatusR. tarasevichiae” was characterized by the gltAand ompBgenes. We report the first case of mixed R. sibiricaand “CandidatusR. tarasevichiae” human infection with a fatal outcome in Russia.

Ixodes persulcatusand Ixodes pavlovskyiticks, two closely related species of the I. ricinus - I. persulcatusgroup, are widely distributed in the southern part of Western Siberia. Recently, the existence of natural hybrids of I. persulcatusand I. pavlovskyiticks has been demonstrated. The aim of this study was to evaluate the abundance of I. persulcatus/pavlovskyihybrids in several locations with different ratios of parental tick species and to investigate the prevalence and genetic variability of a wide range of infectious agents in these hybrids compared to the parental tick species. Natural hybrids of I. persulcatusand I. pavlovskyiticks were identified in all examined locations in Altai and Novosibirsk, Western Siberia, Russia. The abundance of hybrids varied from 7% to 40% in different locations and was maximal in a location with similar proportions of I. persulcatusand I. pavlovskyiticks. For the first time, it was shown that hybrids can be infected with the same agents as their parental tick species: tick-borne encephalitis and Kemerovo viruses, Borrelia afzelii, Borrelia bavariensis, Borrelia garinii, Borrelia miyamotoi, Rickettsia helvetica, Rickettsia raoultii, Rickettsia sibirica, “CandidatusRickettsia tarasevichiae”, Anaplasma phagocytophilum, Ehrlichia muris, “CandidatusNeoehrlichia mikurensis”, and Babesia microti.The prevalence of most bacterial agents in hybrids was intermediate compared to their parental tick species. Most genetic variants of the identified agents have been previously found in the parental tick species. Wide distribution of I. persulcatus/pavlovskyinatural hybrids implies that I. persulcatus, I. pavlovskyiand their hybrids coexist in all I. persulcatus- I. pavlovskyisympatric areas.