Phylogeographical diversity of Anaplasma phagocytophilum in the Asian part of Russia based on multilocus sequence typing and analysis of the ankA gene.

Anaplasma phagocytophilum is a tick-transmitted bacterium that replicates in neutrophil granulocytes and elicits febrile disease in humans and animals; it is widely distributed in the Americas, Europe, Africa, and Asia. A. phagocytophilum is commonly regarded as a single species, but several genetic variants with distinct host distribution and geographical origin have been described. In a previous study, we used multilocus sequence typing (MLST) to characterize 25 A. phagocytophilum strains from Ixodes spp. ticks collected in the Asian part of Russia. The obtained concatenated sequences formed two separate clades reflecting their Asiatic origin and/or the vector species. As one of the clades was related to A. phagocytophilum strains from European voles and shrews, we here extended our analysis to seven samples from the northern red-backed vole Myodes rutilus and included 38 additional strains of Asiatic origin from Ixodes persulcatus, I. pavlovskyi, and their hybrids. Further, the ankA gene was sequenced in 59 A. phagocytophilum strains from ticks and voles. The Russian strains belonged to the two new MLST clusters 5 (38/70) and 6 (32/70), previously referred to as clades within clusters 1 and 3, respectively. The total number of sequence types (STs) found was 27 including 12 new STs. The ankA sequences were unique and formed two new clusters: cluster 8 (34/59) and cluster 10 (25/59). The concordance between MLST and ankA-based typing was 100%. This means that at least two distinct genetic groups of A. phagocytophilum circulate in the Asian Part of Russia whose reservoir hosts and transmission cycles have to be further elucidated.

Asian isolates of Anaplasma phagocytophilum: Multilocus sequence typing.

Anaplasma phagocytophilum is the bacterial agent of granulocytic anaplasmosis in humans and animals; it is widely distributed in Eurasia and North America and transmitted mainly by Ixodes ticks. Several approaches have been used to study genetic diversity in A. phagocytophilum, multilocus sequence typing (MLST) currently being the most reliable and comparable. The MLST method based on seven housekeeping loci, 2877 bp total length, has been used to create and maintain the MLST database available worldwide (https://pubmlst.org/aphagocytophilum/). Before this study, the database contained 150 sequence types (STs) and 418 isolates, 397 of them originating from Europe and 21 from the USA, with none from Asia. We typed 25 A. phagocytophilum isolated from Ixodes ticks collected in the Asian part of Russia and compared the results with the conventional 16S rRNA typing. Substantial variability in the primer binding sites was found, so we had to modify the original primers for six out of seven loci. None of the sequences obtained matched those from the database; 15 new STs and 39 new alleles were revealed. Russian isolates belonged to two clusters, cluster 1 (19 isolates) and 3 (6 isolates), in both of which they formed separate clades. For the first time, we found A. phagocytophilum isolates from Ixodes persulcatus and I. pavlovskyi to belong to cluster 3, previously containing only the strains from voles and shrews. Further research is needed to estimate the prevalence of two MLST clusters of A. phagocytophilum in ticks and vertebrate hosts in Asia.

Bacteria of the Family 'Candidatus Midichloriaceae' in Sympatric Zones of Ixodes Ticks: Genetic Evidence for Vertical Transmission.

Ixodes ticks transmit infectious agents and also harbor their own parasites and symbionts. The presumptive endosymbiont of Ixodes ricinus, 'Candidatus Midichloria mitochondrii', has a unique ability to invade mitochondria within tick ovarian cells and is transovarially transmitted with 100% efficiency. A closely related bacterium, provisionally named Montezuma (now 'Candidatus Lariskella arthropodarum'), was isolated from the Ixodes persulcatus ticks and human blood in 2004 as well as from Ixodes pavlovskyi in 2015. These microorganisms belong to the family 'Candidatus Midichloriaceae fam. nov.' and were detected not only in tick salivary glands, but also in animal blood. Nevertheless, the relative importance of vertical and horizontal routes for their transmission or maintenance in natural tick populations remains unclear. We analyzed the prevalence of L. arthropodarum and M. mitochondrii in two sympatric zones, where I. persulcatus/I. ricinus and I. persulcatus/I. pavlovskyi cohabit and produce interspecific hybrids. A specificity of the associations of L. arthropodarum with I. persulcatus (100%) and M. mitochondrii with I. ricinus (96.2%) was observed in the sympatric zone in Estonia, possibly showing poor contribution of the horizontal route to the overall prevalence of endosymbionts. L. arthropodarum was observed probably multiplying in I. pavlovskyi and also subjected to transovarial transmission, but much less efficiently compared to I. persulcatus. We revealed two new genetic variants of the rrl-rrf intergenic spacer of L. arthropodarum isolated from I. pavlovskyi ticks that possibly could indicate an ongoing process of adaptation of the microorganism to a new host species.

Natural hybridization of the ticks Ixodes persulcatus and Ixodes pavlovskyi in their sympatric populations in Western Siberia.

Hybridization of ticks of the genus Ixodes has been described for several species under laboratory conditions although no molecular genetics evidence confirming interspecific hybridization in nature is available. We have designed a real time PCR targeted on nuclear (ITS2) and mitochondrial (cox1) markers to accurately identify tick species and to detect interspecific hybrids of Ixodes persulcatus and Ixodes pavlovskyi in their sympatric populations in Western Siberia. A survey of 783 individual ticks from a suburb of Tomsk showed that 44.2% of ticks belong to I. pavlovskyi species and 55.8% to I. persulcatus, based on the mtDNA data. Results obtained with the nuclear marker were not consistent, indicating that approximately 10% of the ticks were hybrids and about 5% revealed mtDNA introgression. Both hybridization and introgression have been shown to occur bidirectionally but more efficiently in the mating pair female I. pavlovskyi×male I. persulcatus than vice versa. The existence of the first generation hybrids and backcrosses challenges the existing view about effective reproductive barriers between I. pavlovskyi and I. persulcatus. While using only mitochondrial markers can lead to errors in determining tick species, we propose to use nuclear or both markers instead. The results obtained in the present paper and published earlier suggest that hybridization between closely related tick species in their sympatric zones is common rather than exceptional. The role of hybrid populations of vectors in the evolution of transmitted pathogens is also discussed.

Multilocus spacer analysis revealed highly homogeneous genetic background of Asian type of Borrelia miyamotoi.

Borrelia miyamotoi, a member of the relapsing fever group borreliae, was first isolated in Japan and subsequently found in Ixodes ticks in North America, Europe and Russia. Currently, there are three types of B. miyamotoi: Asian or Siberian (transmitted mainly by Ixodes persulcatus), European (Ixodesricinus) and American (Ixodesscapularis and Ixodespacificus). Despite the great genetic distances between B. miyamotoi types, isolates within a type are characterised by an extremely low genetic variability. In particular, strains of B. miyamotoi of Asian type, isolated in Russia from the Baltic sea to the Far East, have been shown to be identical based on the analysis of several conventional genetic markers, such as 16S rRNA, flagellin, outer membrane protein p66 and glpQ genes. Thus, protein or rRNA - coding genes were shown not to be informative enough in studying genetic diversity of B. miyamotoi within a type. In the present paper, we have attempted to design a new multilocus technique based on eight non-coding intergenic spacers (3686bp in total) and have applied it to the analysis of intra-type genetic variability of В. miyamotoi detected in different regions of Russia and from two tick species, I. persulcatus and Ixodespavlovskyi. However, even though potentially the most variable loci were selected, no genetic variability between studied DNA samples was found, except for one nucleotide substitution in two of them. The sequences obtained were identical to those of the reference strain FR64b. Analysis of the data obtained with the GenBank sequences indicates a highly homogeneous genetic background of B. miyamotoi from the Baltic Sea to the Japanese Islands. In this paper, a hypothesis of clonal expansion of B. miyamotoi is discussed, as well as possible mechanisms for the rapid dissemination of one B. miyamotoi clone over large distances.

Tick-borne encephalitis virus subtypes emerged through rapid vector switches rather than gradual evolution.

Tick-borne encephalitis is the most important human arthropod-borne virus disease in Europe and Russia, with an annual incidence of about 13 thousand people. Tick-borne encephalitis virus (TBEV) is distributed in the natural foci of forest and taiga zones of Eurasia, from the Pacific to the Atlantic coast. Currently, there are three mutually exclusive hypotheses about the origin and distribution of TBEV subtypes, although they are based on the same assumption of gradual evolution. Recently, we have described the structure of TBEV populations in terms of a clusteron approach, a clusteron being a structural unit of viral population [Kovalev and Mukhacheva (2013) Infect. Genet. Evol., 14, 22-28]. This approach allowed us to investigate questions of TBEV evolution in a new way and to propose a hypothesis of quantum evolution due to a vector switch. We also consider a possible mechanism for this switch occurring in interspecific hybrids of ticks. It is necessarily accompanied by a rapid accumulation of mutations in the virus genome, which is contrary to the generally accepted view of gradual evolution in assessing the ages of TBEV populations. The proposed hypothesis could explain and predict not only the formation of new subtypes, but also the emergence of new vector-borne viruses.

Clusterons as a tool for monitoring populations of tick-borne encephalitis virus.

Tick-borne encephalitis (TBE) is a natural focal viral neuroinfection that is widespread in the temperate zone of Eurasia. Knowledge of the genetic structure of tick-borne encephalitis virus (TBEV) populations is important for understanding, not only the origin and evolution of the virus, but also the formation and maintenance of natural foci. A new approach to the differentiation of TBEV strains within subtype, with clusterons as the basis of analysis, has recently been proposed. In the present study, the genetic structure of TBEV-Sib populations has been investigated based on 387 strains isolated in the Middle Urals (Sverdlovsk region). Fourteen of the 18 currently known TBEV-Sib clusterons were identified. They belong to the Asian and Eastern European (Baltic) groups. It was shown that each TBE foci could be characterized by a unique clusteron profile. Three clusterons that emerged within the last 50 years have been identified which implies an active evolutionary process in the TBEV-Sib populations. The greatest diversity of clusterons was observed in the south of the Middle Urals along the Trans-Siberian Way. Such a pattern could reflect the history of colonization of the area and is closely related to the roads passing from Siberia to the European part of Russia through the Urals. In this article, the principles of continuous monitoring in the regional and local TBE foci are proposed, based on the quantitative and qualitative analysis of TBEV-Sib clusteron profiles.

Multilocus sequence analysis of Borrelia burgdorferi s.l. in Russia.

Borrelia burgdorferi sensu lato is a species complex that includes the causative agents of Lyme borreliosis (LB). Classification of the complex was greatly influenced by the method of multilocus sequence typing (MLST), proposed in 2008 and analyzing the spatial distribution of sequence types (STs). Despite the fact that Russia is the largest natural focus of LB to date, it is represented by only 3 strains of the 1323 strains deposited in the MLST database. In this paper, we identified STs for 24 B. burgdorferi s.l. strains isolated from ticks from almost all regions of Russia, 16 of which have not been described so far. It has been shown that the Russian isolates of B. afzelii are of Asian origin and are characterized by a lack of territorial mixing of STs. In contrast, B. garinii and B. bavariensis showed ST mixing between different localities. Comparison of MLST data with the results of sequence analysis of rrf-rrl intergenic spacer led to the conclusion that the previously described genomic groups of B. garinii correspond to the genospecies according to the new classification: 20047 corresponds to B. garinii, and NT29 corresponds to B. bavariensis. The genomic group, ChY13p, characterized by an unusual PCR-RFLP profile, belongs to the species B. garinii (prototype strain 20047). Thus, the use of a reliable method to study the phylogeny and evolution of Borrelia based on MLST, helps to clarify the existing classification. The standardized research procedure and database created could become the basis for a global scientific cooperation in LB research.

Clusteron structure of tick-borne encephalitis virus populations.

Tick-borne encephalitis is a natural focal transmissible zooanthroponosis. The causative agent of the disease is a tick-borne encephalitis virus (TBEV) belonging to the genus Flavivirus of the family Flaviviridae and is widespread in Eurasia. Current TBEV classification based on molecular genetic data comprises three phylogenetically separate subtypes: Far Eastern, European and Siberian (TBEV-Sib). Further differentiation of TBEV isn't developed, making it difficult to investigate the origins, distribution and evolution of the virus. In the present study we determined the nucleotide sequence of the gene E fragment for 282 TBEV-Sib isolates from Ixodes persulcatus ticks or their pools from various natural foci in Russia. Analysis of these sequences and sequences obtained from the GenBank database (more than 600), made it possible to cluster TBEV-Sib strains by identical amino acid sequences of a glycoprotein E fragment. In total, 18 groups were identified (from 3 to 285 strains in the group). It was shown that TBEV strains belonging to the same group are phylogenetically related and have a territorial attachment showing either a local or a corridor type distribution. These groups were named as clusterons showed to be the smallest unit of TBEV classification. The grouping of TBEV strains allows characterization of endemic areas both in quantitative and qualitative composition of the clusterons. The approach could be successfully used to record and monitor the TBEV populations.

Phylogeographical structure of the tick Ixodes persulcatus: a novel view.

The tick Ixodes persulcatus Schulze, 1930, has a wide distribution from the Baltic to the Far East and is a vector of a number of human pathogens. Thus, the study of the genetic structure and evolution of this species is of great epidemiological importance. rRNA genes were used as genetic markers to identify the phylogeographical structure of the ticks. The sequences of gene fragments of 28S (expansion segment D3) and mitochondrial 12S rRNA for 25 and 76 ticks, respectively, that had been collected in various regions of Russia in 2007-2011, were obtained. The sequences of the 28S rRNA D3 segment were identical for all ticks within the studied area. Analysis of the sequences of the mitochondrial 12S rRNA fragment revealed 4 haplotypes with one occurring at a frequency of 0.96. It is shown that the 'deep' population structure of I. persulcatus (McLain et al., 2001) was erroneous because of the inclusion of contaminating fungi sequences of 28S rRNA in the phylogenetic analysis. This was, possibly, due to the use of universal PCR primers that amplify the DNA of a wide range of eukaryotes, particularly of fungi which are common in samples of ticks. The influence of PCR conditions on the preferential amplification of the DNA of different organisms is also demonstrated.