V. V. Panov

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.

The study of heterogeneity of 16S rRNA gene and groESL operone in DNA samples of Anaplasma phagocytophilum, Ehrlichia muris, and “Candidatus Neoehrlichia mikurensis” determined in Ixodes persulcatus ticks on the territory of Ural, Siberia and Far East of Russia

A total of 3552 Ixodes persulcatus from Sverdlovsk, Chelyabinsk, Novosibirsk, Irkutsk regions and Khabarovsk Territory were examined on the Ehrlichia and Anaplasma presence by nested PCR based on the 16S rRNA gene. Both Anaplasma phagocytophilum and Ehrlichia muris DNA were found in I. persulcatus in all studied regions — A. phagocytophilum was detected in 1.3–6.3% of ticks and E. muris — in 2.0–14.1% of ticks. Moreover, “Candidatus Neoehrlichia mikurensis” DNA was found in 8 ticks collected in Novosibirsk, Irkutsk Regions and Khabarovsk Territory. Partial nucleotide sequences of 16S rRNA gene and groESL operone (1240–1300 bp) were determined for 65 samples of A. phagocytophilum, 17 samples of E. muris and 4 samples of “Candidatus Neoehrlichia mikurensis”. Nucleotide sequences of 16S rRNA gene and groESL operone of E. muris and “Candidatus Neoehrlichia mikurensis” were shown to be highly conservative, and nucleotide sequences of groESL operone of both E. muris and “Candidatus Neoehrlichia mikurensis” differed from the sequences found previously in other species of Ixodid tick. On the basis of analysis of the 16S rRNA gene and groESL operone sequences it was concluded that all revealed samples A. phagocytophilum could be divided into 2 groups. GroESL operone sequences of A. phagocytophilum from the first group were identical to each other but significantly differed from the known groESL operone sequences (less than 98.2% of similarity), whereas their 16S rRNA gene sequences were identical to the sequence of widely distributed and pathogenic for human A. phagocytophilum genetic variant (CAHU-HGE1, GenBank AF093788) or differed from it by a single nucleotide substitution. The nucleotide sequences of groESL operone of A. phagocytophilum from the second group differed from each other by 1–4 nucleotides and were closely related (99.2–99.4% of similarity) to the sequences of groESL operone of A. phagocytophilum isolates found in Europe in Ixodes ricinus and roe deer. The nucleotide sequences of the 16S rRNA gene of A. phagocytophilum from the second group were most similar to the sequence of the rare A. phagocytophilum genetic variant previously found only in China (GenBank DQ342324).

Genetic features of DNA of Borrelia miyamotoi transmitted by Ixodes persulcatus

Abstract16S rRNA, p66 and glpQ gene fragments in Borrelia miyamotoi transmitted by Ixodes persulcatus are determined and analyzed. Specific nucleotide sequences of every locus have been shown to be identical. The highest homology level for nucleotide sequences of three loci has been shown for the sequences of strains isolated earlier in Japan. An analysis of the P66 protein amino-acid sequence has shown that the locus corresponding to the surface-exposed domain differs considerably from both Borrelia hermsii, a typical member of tick-borne relapsing fever and Borrelia lonestari, the closest related species. Three genetic variants of Borrelia miyamotoi P66 protein is characterized not only by amino-acid substitutions, but also by deletions.

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

BackgroundThe 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.ResultsThis 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.ConclusionAlmost 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.

First detection of Kemerovo virus in Ixodes pavlovskyi and Ixodes persulcatus ticks collected in Novosibirsk region, Russia.

Kemerovo group viruses are tick-transmitted members of Orbivirus genus of the Reoviridae family that can cause infections of the central nervous system of humans. In this work, Kemerovo virus (KEMV) RNA was detected for the first time in Novosibirsk region of Western Siberia, Russia, in Ixodes pavlovskyi and Ixodes persulcatus ticks.

Distribution of the Ticks Ixodes persulcatus and Ixodes pavlovskyi on the Boundary of the Forest and Forest-Steppe Zones in the Ob Region

Surveys of ixodoid ticks were performed in Novosibirsk Province (Novosibirsk and Toguchin Districts) and in the vicinity of Akademgorodok (Novosibirsk) in 2009–2010. The abundance and distribution of ticks were assessed in 8 types of habitats. Ixodes persulcatus (Schulze, 1930) was collected by flagging in Novosibirsk and Toguchin Districts, with the highest densities of 19 ind./km being observed in habitats with small-leaved trees. Three species of ticks: Ixodes persulcatus, I. pavlovskyi (subspecies I. pavlovskyi occidentalis Filip. et Pan., 1998), and Dermacentor reticulatus (Fabricius, 1794) were recorded in a recreational forest of Akademgorodok. A high abundance (22 ind./km) of I. pavlovskyi was observed in pine forests subjected to considerable recreational load. The abundance of I. persulcatus was the highest in aspen-birch and birch-aspen forests. D. reticulatus was captured in pine forests and fallow lands, its abundance varying from 0.2 to 2 ind./km.

Occurrence and genetic variability of Kemerovo virus in Ixodes ticks from different regions of Western Siberia, Russia and Kazakhstan.

Kemerovo virus (KEMV), a member of the Reoviridae family, Orbivirus genus, is transmitted by Ixodes ticks and can cause aseptic meningitis and meningoencephalitis. Recently, this virus was observed in certain provinces of European part of Russia, Ural, and Western and Eastern Siberia. However, the occurrence and genetic diversity of KEMV in Western Siberia remain poorly studied. Therefore, the aim of this work was to investigate the prevalence and genetic variability of KEMV in Ixodes ticks from Western Siberia. A total of 1958 Ixodes persulcatus, I. pavlovskyi ticks and their hybrids from Novosibirsk and Omsk provinces, Altai Republic (Russia) and East Kazakhstan province (Kazakhstan) were analyzed for the presence of KEMV and tick-borne encephalitis virus (TBEV) RNA. It was observed that the KEMV distribution area in Western Siberia was wider than originally thought and included Northern and Northeastern Altai in addition to the Omsk and Novosibirsk provinces. For the first time, this virus was found in Kazakhstan. The occurrence of KEMV was statistically lower than TBEV in most locations in Western Siberia. KEMV was found both in I. persulcatus and I. pavlovskyi ticks and in their hybrids. Notably, KEMV variants observed in the 2010s were genetically different from those isolated in the 1960s, which indicated the ongoing process of evolution of the Kemerovo virus group. Moreover, the possibility of reassortment for KEMV was demonstrated for the first time.

Infestation of taiga ticks with borrelias in the territory of Novosibirsk Scientific Center (Siberian Branch, Russian Academy of Sciences)

Borrelia specimens were revealed in taiga ticks Ixodes persulcatus collected in the wild by flagging and also in ticks provided by the Vaccination section of the Novosibirsk Scientific Center, Siberian Branch of the Russian Academy of Sciences (NSC); these ticks were obtained from patients attacked by ticks. Isolation of borrelias in the BSK-H medium had demonstrated the presence of B. garinii, B. afzelii, and B. miyamotoi in the territory of NCS. B. miyamotoi isolates were unstable, loosing their growth ability during subsequent cultivation. DNA of the three above species was detected by PCR in tick samples collected by flagging and obtained from humans. DNA of B. garinii was recorded in ticks more often; DNA of B. afzelii was found less frequently; B. miyamotoi DNA was detected in the smallest number of ticks. In ticks collected by flagging, DNA of B. garinii, B. afzelii, and B. miyamotoi was detected in 38.6%, 9.9%, and in 3.9% of specimens, respectively. In ticks collected from attacked humans, the number of positive tests was lower; e.g., DNA of B. garinii, B. afzelii, and B. miyamotoi was detected in 24.2%, 6.9%, and in 5.6% of samples, respectively. Mixed infection of ticks with two Borrelia species was also detected; DNA of B. miyamotoi and of B. garinii was detected in mixed infections more frequently.

Detection of Babesia DNA in small mammals and ixodid ticks in the North Urals, Western Siberia, and Far East of Russia

A total 932 small mammals and 458 questing adult Ixodes persulcatus from Sverdlovsk, Novosibirsk regions and Khabarovsk Territory as well as 128 Haemaphysalis japonica, 34 H. concinna and 29 Dermacentor silvarum from Khabarovsk Territory was examined on the Babesia presence by nested PCR based on the 18S rRNA gene. Babesia microti DNA were found in samples from small mammals in all studied regions — in 36.2% of samples from Sverdlovsk region, in 5.3% of samples from Novosibirsk region and in 6.7% of samples from Khabarovsk territory. The determined B. microti 18S rRNA gene sequences from Novosibirsk region (6 sequences) and from Khabarovsk Territory (10 sequences) were identical to each other and to the sequences of pathogenic for human B. microti US-type, while the determined B. microti 18S rRNA gene sequences from Sverdlovsk region (12 sequences) were identical to the sequence of B. microti strain Munich. B. microti were found most frequently in samples from Myodes spp., they were found also in Microtus spp., Apodemus spp., Sorex spp. and Sicista betulina. One from 347 analyzed I. persulcatus from Novosibirsk region and one from 77 I. persulcatus from Khabarovsk Territory were shown to contain B. microti US-type DNA. One I. persulcatus from Novosibirsk region have contained B. divergens DNA. This is the first determination of B. divergens in I. persulcatus and the first determination of B. microti in I. persulcatus in Asian part of Russia. Three novel genetic variants of Babesia sensu stricto were revealed in three H. japonica from Khabarovsk Territory. One novel Babesia genetic variant was closely related to Babesia sp. revealed in a feral raccoon in Japan (99.9% similarity on the basis of 18S rRNA gene sequences). Two others Babesia genetic variants were most similar to Babesia crassa (97.1–97.6% similarity); they clustered together with ruminant pathogens B. crassa and Babesia major.