Prevalence of Borrelia burgdorferi sensu lato and Borrelia miyamotoi in ixodid ticks in the Far East of Russia.

Borrelia burgdorferi sensu lato (s.l.) DNA was detected by PCR in Ixodes persulcatus Schulze, 1930, Haemaphysalis concinna Koch, 1844, Haemaphysalis japonica douglasi Nuttall et Warburton, 1915 and Dermacentor silvarum Olenev, 1932 ticks collected in the Amur region, the Jewish Autonomous region, the Sakhalin region and on the Khabarovsk territory. Infection rate of I. persulcatus with B. burgdorferi s.l. 10-69% exceeded the corresponding values of three other tick species in all examined regions during 1999-2014 despite different tick abundance and dominance structure. Bacterial loads estimated on the base of quantitative real time PCR varied from 102 to 109 genome-equivalents per a tick with maximal values for I. persulcatus and H. japonica. Phylogenetic analysis of 16S rRNA gene and 5S-23S rRNA intergenic spacer nucleotide sequences revealed two species: 1) Borrelia garinii of Asian type NT29 with several isolates of European type 20047; 2) Borrelia afzelii with identical sequences of the majority of studied isolates and VS461 reference strain in all regions except the Sakhalin Island where B. afzelii was not found. Borrelia miyamotoi of the relapsing fever group was detected as monoinfection or in combination with B. burgdorferi s.l. in 4.0 ±â€¯0.9% and 4.8 ±â€¯0.9% I. persulcatus ticks, respectively. Multiple locus sequence analysis of three fragments of 16S rRNA, glpQ and p66 genes proved that all the Far Eastern B. miyamotoi isolates belonged to the Asian type identical to FR64b strain (GenBank CP004217) from Japan. Wide distribution of Borrelia DNA in ticks, relative genetic homogeneity with similar sequences of the coding regions and the intergenic spacer of Borrelia wild isolates and temporal stability with high homology levels of the Far Eastern isolates of B. garinii, B. afzelii and B. miyamotoi with previously described spirochetes from the surrounding regions of Russia, China and Japan allowed us to suggest multiple ecological niches as the stability factor of the parasitic system.

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.

Tick-borne encephalitis virus in arthropod vectors in the Far East of Russia.

Isolates of tick-borne encephalitis virus (TBEV) from arthropod vectors (ticks and mosquitoes) in the Amur, the Jewish Autonomous and the Sakhalin regions as well as on the Khabarovsk territory of the Far East of Russia were studied. Different proportions of four main tick species of the family Ixodidae: Ixodes persulcatus P. Schulze, 1930; Haemaphysalis concinna Koch, 1844; Haemaphysalis japonica douglasi Nuttall et Warburton, 1915 and Dermacentor silvarum Olenev, 1932 were found in forests and near settlements. RT-PCR of TBEV RNA in adult ticks collected from vegetation in 1999-2014 revealed average infection rates of 7.9 ±â€¯0.7% in I. persulcatus, of 5.6 ±â€¯1.0% in H. concinna, of 2.0 ±â€¯2.0% in H. japonica, and of 1.3 ±â€¯1.3% in D. silvarum. Viral loads varied in a range from 102 to 109 TBEV genome-equivalents per a tick with the maximal values in I. persulcatus and H. japonica. Molecular typing using reverse transcription with subsequent real time PCR with subtype-specific fluorescent probes demonstrated that the Far Eastern (FE) subtype of TBEV predominated both in mono-infections and in mixed infection with the Siberian (Sib) subtype in I. persulcatus pools. TBEV strains of the FE subtype were isolated from I. persulcatus, H. concinna and from a pool of Aedes vexans mosquitoes. Ten TBEV strains isolated from I. persulcatus from the Khabarovsk territory and the Jewish Autonomous region between 1985 and 2013 cluster with the TBEV vaccine strain Sofjin of the FE subtype isolated from human brain in 1937. A TBEV strain from H. concinna collected in the Amur region (GenBank accession number KF880803) is similar to the vaccine strain 205 isolated in 1973 from I. persulcatus collected in the Jewish Autonomous region. The TBEV strain Lazo MP36 of the FE subtype isolated from a pool of A. vexans in the Khabarovsk territory in 2014 (KT001073) differs from strains isolated from 1) I. persulcatus (including the vaccine strain 205) and H. concinna; 2) mosquitoes [strain Malishevo (KJ744034) isolated in 1978 from Aedes vexans nipponii in the Khabarovsk territory]; and 3) human brain (including the vaccine strain Sofjin). Accordingly, in the far eastern natural foci, TBEV of the prevailing FE subtype has remained stable since 1937. Both Russian vaccines against TBE based on the FE strains (Sofjin and 205) are similar to the new viral isolates and might protect against infection.

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.

Genetic diversity of Anaplasma and Ehrlichia in the Asian part of Russia.

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.

Molecular screening of Bartonella species in rodents from the Russian Far East.

A potentially broad-range PCR system for gltA gene amplification has been designed. A nearly full (992 bp) sequence of the gltA gene was amplified in two steps. DNA was extracted from organs (spleen and liver) of four mostly spread species of rodents: Apodemus agrarius and Apodemus peninsulae mice; Clethrionomys rufocanus and Microtus fortis voles. All amplicons have been sequenced. The incidence of Bartonella species in rodents was 73%, 60%, 60%, and 83% for the above-mentioned rodents, respectively. Several genotypes have been identified, including two gltA genotypes close to B. taylorii, four gltA genotypes close to B. grahamii, and one genotype of a potentially new species. Several Bartonella species were found in both mice species, although each vole species harbored a single species of Bartonella. High incidence of Bartonella infection was found in all studied species of small rodents in the Russian Far East. Several amplified genotypes probably belong to local strains of B. taylorii and B. grahamii, and one genotype represents a possible new species.