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.

"Candidatus Rickettsia uralica" and "Candidatus Rickettsia thierseensis" are genetic variants of one species.

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.

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.

A fatal case of tick-borne rickettsiosis caused by mixed Rickettsia sibirica subsp. sibirica and "Candidatus Rickettsia tarasevichiae" infection in Russia.

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.

Ixodes persulcatus/pavlovskyi natural hybrids in Siberia: Occurrence in sympatric areas and infection by a wide range of tick-transmitted agents.

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.

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.

Detection and genetic characterization of a wide range of infectious agents in Ixodes pavlovskyi ticks in Western Siberia, Russia.

BACKGROUND: The Ixodes pavlovskyi tick species, a member of the I. persulcatus/I. ricinus group, was discovered in the middle of the 20th century in the Russian Far East. Limited data have been reported on the detection of infectious agents in this tick species. The aim of this study was to investigate the prevalence and genetic variability of a wide range of infectious agents in I. pavlovskyi ticks collected in their traditional and recently invaded habitats, the Altai Mountains and Novosibirsk Province, respectively, which are both located within the Western Siberian part of the I. pavlovskyi distribution area. RESULTS: This study reports the novel discovery of Borrelia bavariensis, Rickettsia helvetica, R. heilongjiangensis, R. raoultii, "Candidatus Rickettsia tarasevichiae", Anaplasma phagocytophilum, Ehrlichia muris, "Candidatus Neoehrlichia mikurensis" and Babesia microti in I. pavlovskyi ticks. In addition, we confirmed the previous identification of B. afzelii, B. garinii and B. miyamotoi, as well as tick-borne encephalitis and Kemerovo viruses in this tick species. The prevalence and some genetic characteristics of all of the tested agents were compared with those found in I. persulcatus ticks that were collected at the same time in the same locations, where these tick species occur in sympatry. It was shown that the prevalence and genotypes of many of the identified pathogens did not significantly differ between I. pavlovskyi and I. persulcatus ticks. However, I. pavlovskyi ticks were significantly more often infected by B. garinii and less often by B. bavariensis, B. afzelii, "Ca. R. tarasevichiae", and E. muris than I. persulcatus ticks in both studied regions. Moreover, new genetic variants of B. burgdorferi (sensu lato) and Rickettsia spp. as well as tick-borne encephalitis and Kemerovo viruses were found in both I. pavlovskyi and I. persulcatus ticks. CONCLUSION: Almost all pathogens that were previously detected in I. persulcatus ticks were identified in I. pavlovskyi ticks; however, the distribution of species belonging to the B. burgdorferi (sensu lato) complex, the genus Rickettsia, and the family Anaplasmataceae was different between the two tick species. Several new genetic variants of viral and bacterial agents were identified in I. pavlovskyi and I. persulcatus ticks.

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.