Novel Genetic Lineages of Rickettsia helvetica Associated with Ixodes apronophorus and Ixodes trianguliceps Ticks.

Ixodes apronophorus is an insufficiently studied nidicolous tick species. For the first time, the prevalence and genetic diversity of Rickettsia spp. in Ixodes apronophorus, Ixodes persulcatus, and Ixodes trianguliceps ticks from their sympatric habitats in Western Siberia were investigated. Rickettsia helvetica was first identified in I. apronophorus with a prevalence exceeding 60%. "Candidatus Rickettsia tarasevichiae" dominated in I. persulcatus, whereas I. trianguliceps were infected with "Candidatus Rickettsia uralica", R. helvetica, and "Ca. R. tarasevichiae". For larvae collected from small mammals, a strong association was observed between tick species and rickettsiae species/sequence variants, indicating that co-feeding transmission in studied habitats is absent or its impact is insignificant. Phylogenetic analysis of all available R. helvetica sequences demonstrated the presence of four distinct genetic lineages. Most sequences from I. apronophorus belong to the unique lineage III, and single sequences cluster into the lineage I alongside sequences from European I. ricinus and Siberian I. persulcatus. Rickettsia helvetica sequences from I. trianguliceps, along with sequences from I. persulcatus from northwestern Russia, form lineage II. Other known R. helvetica sequences from I. persulcatus from the Far East group into the lineage IV. The obtained results demonstrated the high genetic variability of R. helvetica.

Multilocus genetic analysis indicates taxonomic status of "Candidatus Rickettsia mendelii" as a separate basal group.

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.

Occurrence and clinical manifestations of tick-borne rickettsioses in Western Siberia: First Russian cases of Rickettsia aeschlimannii and Rickettsia slovaca infections.

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.

Genetic variability of Anaplasmataceae circulating in small mammals and ticks in an Ixodes persulcatus/Ixodes trianguliceps sympatric area in Russian Siberia.

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.

Long-term persistence of Anaplasma phagocytophilum and Ehrlichia muris in wild rodents.

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).

Genetic and morphological characterization of Ixodes apronophorus from Western Siberia, Russia.

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.

Genetic variability of Rickettsia spp. in Dermacentor and Haemaphysalis ticks from the Russian Far East.

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.

High prevalence of Babesia microti 'Munich' type in small mammals from an Ixodes persulcatus/Ixodes trianguliceps sympatric area in the Omsk region, Russia.

Babesia microti is a genetically diverse group of protozoan parasites whose life cycle is associated with both small mammals and Ixodes spp. ticks. In this study, the prevalence of different B. microti genetic groups in ticks and small rodents in an area with Ixodes persulcatus and Ixodes trianguliceps occurring in sympatry was examined. A total of 541 small mammals were captured during eight sampling periods between 2013 and 2015 at a site in the Omsk region of Russia and tested for the presence of B. microti using nested PCR with subsequent sequencing of positive samples. B. microti DNA was found in 31.6 % of examined samples, and prevalence rates ranged from 5.3 to 61.6 % in different sampling periods. The sequenced B. microti samples belonged to two genetic groups: enzootic B. microti 'Munich' type and zoonotic B. microti 'US' type. B. microti 'Munich' type was more common across all sampling periods, with greater than 80 % prevalence in infected animals. Despite the high B. microti 'Munich'-type prevalence in voles, B. microti was not found in any of 394 adult I. persulcatus ticks collected by flagging or in the 84 I. persulcatus or 20 I. trianguliceps ticks taken from voles and molted under laboratory conditions. It was demonstrated that B. microti 'Munich'-type DNA can be detected in the blood of naturally infected voles for at least 20 weeks after capture. Thus, the high prevalence of B. microti 'Munich' type in small mammals may be explained by the prolonged persistence of B. microti in the blood of wild voles.

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.

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.