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To date, the phylogeny of Rickettsia spp. from basal groups is based on the small number of identified species. Thus, the finding of "Candidatus Rickettsia mendelii" in 2016 is of great interest. In this study, "Ca. R. mendelii" was first identified in the Asian region in a new carrier, Ixodes pavlovskyi. "Candidatus R. mendelii", along with "Candidatus Rickettsia tarasevichiae", were found in Ixodes ticks collected on Russky Island (the Far East), where I. pavlovskyi coexists with I. persulcatus. To establish the taxonomic position of "Ca. R. mendelii", a detailed genetic study was carried out. "Candidatus R. mendelii" was genotyped by five genetic fragments (16S rRNA, gltA, and ompB genes, groESL operon, and 23S-5S IGS region); among them, the ompB gene, groESL operon and 23S-5S IGS region were sequenced for the first time. In addition, "Ca. R. tarasevichiae" was genetically characterized by eight genetic loci (16S rRNA, gltA, ompA, ompB, sca4, htrA genes, groESL operon, and 23S-5S IGS region), of which the sca4 gene was first determined. Phylogenetic analysis indicated that regardless of analyzed genetic loci, "Ca. R. mendelii" formed a separate well-supported cluster on each phylogenetic tree. Phylogenetic analysis based on concatenated sequences of gltA, ompB, and groEL gene fragments (total length of 3191 bp) demonstrated that "Ca. R. mendelii", like Rickettsia bellii, is a basal group of Rickettsia., (Copyright © 2022. Published by Elsevier GmbH.)

Four genospecies from the Borrelia burgdorferi sensu lato complex were detected in Ixodes persulcatus and Ixodes pavlovskyi ticks from Siberia and genetically characterized. The presence of Borrelia spp. in Ixodes apronophorus and Ixodes trianguliceps ticks found in Asia has never been studied. In this study, genetic diversity of B. burgdorferi s.l. was investigated in three I. persulcatus / I. trianguliceps / I. apronophorus sympatric habitats in Western Siberia. Three groups of samples were examined: (i) ticks that were taken from rodents and molted in a laboratory; (ii) non-molted ticks collected from rodents; (iii) specimens from small mammals. Expectedly, Borrelia afzelii and Borrelia bavariensis were detected in I. persulcatus and in small mammals from the studied locations. Borrelia bavariensis was first found in molted I. apronophorus and I. trianguliceps. Identical genovariants of B. bavariensis were found in I. apronophorus, I. trianguliceps, and I. persulcatus. In addition, a new Borrelia genovariant was discovered in non-molted and molted I. apronophorus and non-molted I. persulcatus and I. trianguliceps, as well as in small mammals. This new genovariant was genetically characterized using MLST and single locus sequence analysis, which indicated that the new Borrelia genovariant significantly differs from all known Borrelia species. We propose the name "Candidatus Borrelia sibirica" for this putative new species., (Copyright © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The spotted fever group is the largest group in the Rickettsia genus, and new candidate species of this group are found throughout the world. "Candidatus Rickettsia thierseensis" was observed in a single Ixodes ricinus in Austria in 2020. However, sequences of five genes (total length: 5305 bp) of the "Candidatus R. thierseensis" were nearly identical to those available for "Candidatus Rickettsia uralica" identified in Ixodes trianguliceps and small mammals in Russia in 2015. To confirm the identity of these species, appropriate fragments of seven genetic loci from four isolates of "Candidatus R. uralica" were sequenced and compared with the "Candidatus R. thierseensis" sequences. Additionally, the groEL gene fragment was first sequenced for "Candidatus R. uralica". In this analysis, "Candidatus R. uralica" and "Candidatus R. thierseensis" demonstrated 100% identity in the 23S-5S IGS region (354 bp) and in fragments of the 16S rRNA (991 bp), gltA (1119 bp), and htrA (463 bp) genes, differing by only one substitution in each of the ompA (3696 bp), ompB (4871 bp), and sca4 (2971 bp) gene fragments. Thus, "Candidatus R. uralica" and "Candidatus R. thierseensis" are likely the same species based on comparative sequence analysis., (Copyright © 2022. Published by Elsevier GmbH.)

Rickettsia sibirica subsp. sibirica is a main agent of tick-borne rickettsioses in Western Siberia, Russia. Recently, the first cases of Rickettsia raoultii infection in patients hospitalized in Novosibirsk Province were described. The aim of this study was to establish the etiologic agents of tick-borne rickettsioses in Western Siberia during three epidemiological seasons. Clinical samples from 1008 patients hospitalized after tick bites in April-September 2017-2019 were examined by nested PCR for the presence of Rickettsia spp. All positive specimens were genetically characterized by sequencing of the gltA gene; some specimens were also genotyped based on the 16S rRNA, ompA, and ompB genes. Rickettsia spp. DNA was detected in clinical samples from 56 (5.6%) patients. Five Rickettsia species were identified: R. sibirica (n = 28), R. raoultii (n = 15), Rickettsia aeschlimannii (n = 3), "Candidatus Rickettsia tarasevichiae" (n = 2), and Rickettsia slovaca (n = 1). In addition, new unclassified Rickettsia genovariants were found in specimens from seven patients. Patients with R. raoultii infection presented rash, eschar, and high serum aminotransferase levels less frequently compared to patients with R. sibirica infections, but more frequently showed neurological symptoms. Among other patients, only persons with an R. aeschlimannii infection had rash and/or eschar, which are typical for tick-borne rickettsioses. The current study showed that R. raoultii is a common agent of tick-borne rickettsioses in Novosibirsk Province. DNA from R. aeschlimannii and R. slovaca was found in clinical samples of patients in the Russian Federation for the first time., (Copyright © 2022. Published by Elsevier GmbH.)

A total of 705 rodents from Myodes, Microtus, and Apodemus genera, 396 adult questing Ixodes persulcatus, and 115 Ixodes larvae and nymphs taken from rodents (and then molted under laboratory conditions to nymphs and adults) were collected in 2013-2018 in Omsk province, Russian Siberia, and examined for the presence of Anaplasmataceae. DNA of Anaplasma phagocytophilum was detected in 29.5 % rodents, 3.8 % questing I. persulcatus, two molted adult I. persulcatus, and one molted adult Ixodes trianguliceps. Ehrlichia muris DNA was found in specimens from 12.1 % rodents, 3.0 % questing I. persulcatus, 14 % molted adult I. persulcatus, and one molted adult I. trianguliceps. Neoehrlichia mikurensis DNA was found in 0.6 % blood samples. It was suggested that in the studied area A. phagocytophilum and E. muris are mainly transmitted to small rodents by I. trianguliceps and I. persulcatus, respectively. Based on groEL gene sequence analysis, three phylogenetic clusters of A. phagocytophilum (clusters 4, 5, 6, according to Jaarsma et al., 2019) were identified. Most of genotyped A. phagocytophilum isolates obtained from rodents (87.6 %) and a single isolate found in a molted adult I. trianguliceps belonged to cluster 5. Cluster 6 contained 11.8 % genotyped specimens from rodents, and one questing and two molted adult I. persulcatus, while cluster 4 included specimens from 93 % genotyped questing I. persulcatus and one vole. The finding of A. phagocytophilum from clusters 5 and 6 in voles from the same sampling area indicated that clusters 5 and 6 segregate according to the tick-carriers, but not the geography. Most of the genotyped specimens of E. muris and N. mikurensis corresponded to typical genotypes detected in Asian Russia previously. In addition, new genetic variants of E. muris and N. mikurensis, which significantly differed from other known isolates and formed separate branches on phylogenetic trees, were identified in several voles., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

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 Myodes spp. 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 "Candidatus Neoehrlichia mikurensis". Anaplasma phagocytophilum, E. muris, and mixed infections were found in 29 (49 %), 17 (29 %), and 14 (24 %) voles, respectively. DNA of "Candidatus N. mikurensis" was not identified. Long-term persistence of A. phagocytophilum and E. muris in naturally infected Myodes spp. was shown for the first time. Thus, 12 animals with A. phagocytophilum were 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. muris for 26-40 weeks. Fragments of groESL operon were sequenced for genotyping A. phagocytophilum and 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 trianguliceps ticks from Western Siberia and European countries; one sequence was previously identified in rodents and Ixodes persulcatus and Ixodes pavlovskyi ticks from Asian Russia. Sequences of groESL fragments from E. muris were close to those determined previously in small mammals and I. persulcatus ticks from Siberia. In addition, a new groESL sequence of E. muris was identified in one vole and the sequence substantially differed from all known corresponding E. muris sequences (≥ 14 mismatches)., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Genetic variability of I. apronophorus from Western Siberia, Russia was examined using the nuclear internal transcribed spacer 2 (ITS2) and mitochondrial 16S rRNA and cytochrome c oxidase subunit 1 (cox1) genes and compared to those of Ixodes persulcatus and Ixodes trianguliceps from the same site. The I. apronophorus sequences demonstrated the highest nucleotide and haplotype diversity for both mitochondrial genes, whereas I. persulcatus was more variable in the nuclear ITS2. Phylogenetic analysis of the molecular sequence data showed that I. apronophorus differed from other Ixodes species, including Romanian I. apronophorus. The level of identity between 16S rRNA gene sequences of Siberian and Romanian I. apronophorus was only 91%; these sequences did not form a monophyletic group, indicating that I. apronophorus from Siberia and Romania could be different tick species. The analysis of morphological features of the Siberian I. apronophorus confirmed their consistency with those for the previously described I. apronophorus species. Based on the 16S rRNA and ITS2 sequences, Siberian I. apronophorus clustered together with Ixodes kazakstani and Ixodes scapularis, which are the recognized members of the Ixodes ricinus-I. persulcatus species complex within the subgenus Ixodes, and can be assigned to this complex., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Ixodes persulcatus and Ixodes pavlovskyi ticks, two closely related species of the I. ricinus - I. persulcatus group, are widely distributed in the southern part of Western Siberia. Recently, the existence of natural hybrids of I. persulcatus and I. pavlovskyi ticks has been demonstrated. The aim of this study was to evaluate the abundance of I. persulcatus/pavlovskyi hybrids 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. persulcatus and I. pavlovskyi ticks 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. persulcatus and I. pavlovskyi ticks. 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, "Candidatus Rickettsia tarasevichiae", Anaplasma phagocytophilum, Ehrlichia muris, "Candidatus Neoehrlichia 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/pavlovskyi natural hybrids implies that I. persulcatus, I. pavlovskyi and their hybrids coexist in all I. persulcatus - I. pavlovskyi sympatric areas., (Copyright © 2019. Published by Elsevier GmbH.)

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 sibirica subsp. sibirica and "Candidatus Rickettsia tarasevichiae" DNA was identified in both blood and brain samples. Rickettsia sibirica was characterized by the gltA, ompA and ompB genes, and "Candidatus R. tarasevichiae" was characterized by the gltA and ompB genes. We report the first case of mixed R. sibirica and "Candidatus R. tarasevichiae" human infection with a fatal outcome in Russia., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

The Russian Far East is an endemic region for tick-borne rickettsioses. However, the prevalence and genetic variability of Rickettsia species in this region have not been extensively investigated. In this study, 188 Dermacentor silvarum, 439 Haemaphysalis concinna, and 374 Haemaphysalis japonica adult ticks were collected from four locations in Khabarovsk Province and three locations in Amur Province in the Russian Far East. These ticks were examined for the presence of Rickettsia spp. by amplifying a fragment of the gltA gene. Identified rickettsiae were genotyped by sequencing of the gltA, 16S rRNA, ompA, ompB, and sca4 genes. In the examined ticks, Rickettsia heilongjiangensis, the causative agent of Far-Eastern tick-borne rickettsiosis, was found in 10.5% of H. concinna and in 1.9% of H. japonica ticks, while Rickettsia sibirica, the agent of Siberian tick typhus, was detected in only one H. concinna tick. In addition, Rickettsia raoultii was found predominantly in D. silvarum (>70%) and significantly less frequently in Haemaphysalis ticks (<3%). "Candidatus Rickettsia tarasevichiae" was found in all examined tick species (1.6-5.3% in different species). Notably, this study is the first observation of "Candidatus R. tarasevichiae" in D. silvarum ticks. Moreover, DNA of Rickettsia canadensis was found for the first time in a H. japonica tick; DNA of Rickettsia aeschlimannii was revealed for the first time in H. concinna and H. japonica ticks. "Candidatus Rickettsia principis" and "Candidatus Rickettsia rara" were found in Haemaphysalis spp. ticks. "Candidatus R. principis" was associated with H. japonica and identified in 5.6% of the examined ticks, while "Candidatus R. rara" was found more frequently in H. concinna (3.0%) compared to H. japonica ticks (1.1%). In this study, "Candidatus R. principis" and "Candidatus R. rara" were characterized for the first time by the 16S rRNA, ompA, ompB, and sca4 genes., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

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. persulcatus and I. trianguliceps species 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., (Copyright © 2016 Elsevier GmbH. 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.)

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.)