Detection of Candidatus Neoehrlichia mikurensis and Ehrlichia muris in Estonian ticks.

So far neglected bacteria like Candidatud Neoehrlichia mikurensis and Ehrlichia muris-like agents get increased attention in the recent past. Ixodid ticks were demonstrated to harbor both of these pathogens. Estonia is populated by two medically important tick species, I. ricinus and I. persulcatus. In this study the presence of E. muris and Candidatus N. mikurensis in these two tick species was investigated. Tick DNA was analyzed by nested PCR and subsequent sequencing for the presence of 16S rRNA of E. muris and Candidatus N. mikurensis. Positive samples were further confirmed by amplification and sequencing of the partial groESL-operon. The obtained partial groESL sequences were used for construction of a maximum likelihood tree. In total, 776 ticks from 36 collection sites situated in 7 counties on the mainland of Estonia and 2 sites situated in one county on the island Saaremaa were collected. 548 were I. ricinus and 228 were I. persulcatus. Only in 5 counties (11 sites) samples positive for the Anaplasmataceae 16S rRNA gene were found. The percentage of Candidatus N. mikurensis positive ticks varied from 1% to 9.1% at different sites. In Eastern and South-Eastern Estonia, the area where I. ricinus and I. persulcatus are sympatric, no Candidatus N. mikurensis was found. Ticks carrying E. muris were found in three counties, the site-specific percentage of positive ticks varied from 1.2% to 25.6%. This is the first study revealing the presence of Candidatus N. mikurensis and E. muris in Estonian ticks. Candidatus N. mikurensis was found only in the western part of the country exclusively in I. ricinus and the phylogenetic analysis revealed close relatedness of the Estonian sequences to other European Candidatus N. mikurensis strains. E. muris was detected mostly in I. persulcatus and only in one I. ricinus in the sympatric area of both tick species. This is in correspondence with the observation that this pathogen is more often found in I. persulcatus than in I. ricinus. This study demonstrates the presence of Candidatus N. mikurensis and E. muris in Estonian ticks and highlights the necessity to raise awareness of symptoms by healthcare professionals.

Detection and identification of Rickettsia species in Ixodes tick populations from Estonia.

A total of 1640 ticks collected in different geographical parts of Estonia were screened for the presence of Rickettsia species DNA by real-time PCR. DNA of Rickettsia was detected in 83 out of 1640 questing ticks with an overall prevalence of 5.1%. The majority of the ticks infected by rickettsiae were Ixodes ricinus (74 of 83), while 9 of the 83 positive ticks were Ixodes persulcatus. For rickettsial species identification, a part of the citrate synthase gltA gene was sequenced. The majority of the positive samples were identified as Rickettsia helvetica (81 out of 83) and two of the samples were identified as Rickettsia monacensis and Candidatus R. tarasevichiae, respectively. Genetic characterization based on the partial gltA gene showed that the Estonian sequences within the R. helvetica, R. monacensis and Candidatus R. tarasevichiae species demonstrated 100% similarity with sequences deposited in GenBank, originating from Rickettsia species distributed over large territories from Europe to Asia.

Amino Acid Polymorphisms Within the Entire HCV NS5A Region in Estonian Chronic HCV 1b Patients With Different Treatment Response.

BACKGROUND: A substantial proportion of hepatitis C virus (HCV)-1b infected patients do not response to pegylated interferon-α plus ribavirin (PegIFNα/RBV) combination therapy that was partially associated with mutations in the non-structural 5A (NS5A) protein. OBJECTIVES: Analysis of NS5A polymorphisms in HCV genotype 1b pre-treatment serum samples from Estonian patients and their effect on the treatment response. PATIENTS AND METHODS: Twenty-nine complete NS5A sequences obtained from patients with chronic HCV-1b infection who had received combined therapy with PegIFNα-2a/RBV were analyzed and compared with the prototype strain HCV-J. Twelve patients achieved a sustained virological response (SVR), 15 were non-SVR and 2 patients stopped treatment because of side effects. RESULTS: No significant difference in total number of amino acid mutations was observed between isolates from SVR and non-SVR patients in any known regions of the NS5A protein. However, specific amino acid substitutions at positions 1989 and 2283 correlated significantly with SVR, mutations at positions 1979, 2107, 2171 and 2382 were associated with non-response to treatment and amino acid substitution at position 2319 was observed in relapsers. At phylogenetic analysis, NS5A nucleotide sequences have been subdivided into four groups characterized by the different treatment response. Twenty-four novel nucleotide polymorphisms and 11 novel amino acid polymorphisms were identified based on the phylogenetic tree topology. CONCLUSIONS: Specific amino acid substitutions correlating with the treatment response were found. Polymorphisms revealed by phylogenetic analysis may define the signature patterns for treatment susceptible and treatment resistant strains prevalent in Estonia.

Borrelia burgdorferi sensu lato prevalence in tick populations in Estonia.

BACKGROUND: Estonia is located in a unique area of co-distribution of Ixodes ricinus and I. persulcatus, which are the main tick vectors of Borrelia burgdorferi sensu lato. In the last decade, the incidence rate of Lyme borreliosis in Estonia has increased dramatically up to 115.4 per 100,000 in 2012. Here we present the first survey of the presence, the prevalence and genetic characteristics of B. burgdorferi s.l. complex spirochetes in the tick population in Estonia. METHODS: During the years 2006-2009, 2833 unfed Ixodes ricinus and I. persulcatus were collected from 43 sites in 7 counties in mainland Estonia as well as in 10 sites on the Saaremaa Island. DNA samples from ticks were analyzed individually using nested PCR of the ribosomal 5S-23S spacer region followed by bidirectional sequencing. RESULTS: The overall estimated prevalence of B. burgdorferi s.l was 9.7% and varied from 4.9% to 24.2% on the mainland and to 10.7% in Saaremaa Island. Ixodes persulcatus ticks showed significantly higher prevalence rates compared to that in I. ricinus-16.3% and 8.2%, respectively. The most prevalent genospecies was B. afzelii which was detected in 53.5% of Borrelia-positive ticks, followed by B. garinii and B. valaisiana with 26.2% and 5.5%, respectively. Also, B. bavariensis and B. burgdorferi s.s. DNA in single I. ricinus ticks were detected. Borrelia afzelii, B. garinii and B. valaisiana were detected in both tick species. Two genetic subgroups of B. garinii (NT29 and 20047) and two genetic subgroups of B. afzelii (NT28 and VS461) were found to be circulating in all studied regions as well as in both tick species, except B. garinii subgroup NT29, which was found only in I. persulcatus ticks. CONCLUSIONS: In the current study we detected the circulation of five B. burgdorferi s.l. genospecies and estimated the prevalence in ticks in different regions of Estonia. Detection and genetic characterization of Borrelia genospecies, especially those of public health importance, in the natural foci may help assessing high risk areas of human exposure to B. burgdorferi s.l.

Detection and characterization of tick-borne encephalitis virus in Baltic countries and eastern Poland.

Ticks were collected from the vegetation in the Baltic countries Estonia, Latvia, Lithuania and eastern Poland and analyzed for the presence of tick-borne encephalitis virus (TBEV) by amplification of the partial E and NS3 genes. In Estonia we found statistically significant differences in the TBEV prevalence between I. persulcatus and I. ricinus ticks (4.23% and 0.42%, respectively). In Latvia, the difference in TBEV prevalence between the two species was not statistically significant (1.02% for I. persulcatus and 1.51% for I. ricinus, respectively). In Lithuania and Poland TBEV was detected in 0.24% and 0.11% of I. ricinus ticks, respectively. Genetic characterization of the partial E and NS3 sequences demonstrated that the TBEV strains belonged to the European subtype in all countries, as well as to the Siberian subtype in Estonia. We also found that in areas where ranges of two tick species overlap, the TBEV subtypes may be detected not only in their natural vector, but also in sympatric tick species.

Tick-borne pathogens in ticks feeding on migratory passerines in Western part of Estonia.

During southward migration in the years 2006-2009, 178 migratory passerines of 24 bird species infested with ticks were captured at bird stations in Western Estonia. In total, 249 nymphal ticks were removed and analyzed individually for the presence of Borrelia burgdorferi sensu lato (s.l.), tick-borne encephalitis virus (TBEV), and Anaplasma phagocytophilum. The majority of ticks were collected from Acrocephalus (58%), Turdus (13%), Sylvia (8%), and Parus (6%) bird species. Tick-borne pathogens were detected in nymphs removed from Acrocephalus, Turdus, and Parus bird species. TBEV of the European subtype was detected in 1 I. ricinus nymph removed from A. palustris. B. burgdorferi s.l. DNA was found in 11 ticks (4.4%) collected from Turdus and Parus species. Bird-associated B. garinii and B. valaisiana were detected in I. ricinus nymphs removed from T. merula. Rodent-associated B. afzelii was detected in 3 I. ricinus nymphs from 2 P. major birds. One of the B. afzelii-positive nymphs was infected with a mix of 2 B. afzelii strains, whereas 1 of these strains was also detected in another nymph feeding on the same great tit. The sharing of the same B. afzelii strain by 2 nymphs indicates a possible transmission of B. afzelii by co-feeding on a bird. A. phagocytophilum DNA was detected in 1 I. ricinus nymph feeding on a T. iliacus. The results of the study confirm the possible role of migratory birds in the dispersal of ticks infected with tick-borne pathogens along the southward migration route via Estonia.

Detection and genetic characterization of relapsing fever spirochete Borrelia miyamotoi in Estonian ticks.

During the years 2008-2010 I. ricinus and I. persulcatus ticks were collected from 64 sites in mainland Estonia and on the island Saaremaa. Presence of B. miyamotoi was found in 0.9% (23/2622) of ticks. The prevalence in I. persulcatus and I. ricinus ticks differed significantly, 2.7% (15/561) and 0.4% (8/2061), respectively. The highest prevalence rates were in found South-Eastern Estonia in an area of I. persulcatus and I. ricinus sympatry and varied from 1.4% (1/73) to 2.8% (5/178). Co-infections with B. burgdorferi s.l. group spirochetes and tick-borne encephalitis virus were also revealed. Genetic characterization of partial 16S rRNA, p66 and glpQ genes demonstrated that Estonian sequences belong to two types of B. miyamotoi and cluster with sequences from Europe and the European part of Russia, as well as with sequences from Siberia, Asia and Japan, here designated as European and Asian types, respectively. Estonian sequences of the European type were obtained from I. ricinus ticks only, whereas the Asian type of B. miyamotoi was shown for both tick species in the sympatric regions.

Detection and characterization of Babesia species in Ixodes ticks in Estonia.

The presence of Babesia spp. was studied in 2603 Ixodes ricinus and Ixodes persulcatus ticks collected at seven sites in Estonia. By reverse line blot screening, Babesia spp. was detected in 36 (1.4%) ticks, among them 18 (0.7%) were further recognized by a Babesia microti probe, 3 (0.1%) by a Babesia divergens probe, and the other 15 (0.6%) were recognized only by the universal Babesia spp. "catch all" probe. Sequence analyses of 6 of these 15 samples revealed that all of them belonged to Babesia sp. EU1. B. microti was detected in both tick species I. ricinus and I. persulcatus at the seven sites, whereas B. divergens-like and Babesia sp. EU1 were found only in I. persulcatus and I. ricinus, respectively. Genetic characterization based on partial 18S rRNA showed that the Estonian sequences of B. microti, B. divergens-like, and Babesia sp. EU1 share a high rate of similarity and are closely related to sequences from other European countries, Siberia, and United States. The present study demonstrated for the first time the existence and distribution of Babesia spp. in I. persulcatus and I. ricinus ticks in Estonia.

D2: major subgenotype of hepatitis B virus in Russia and the Baltic region.

Complete or almost complete hepatitis B virus (HBV) genomes were sequenced for 13 genotype A and 42 genotype D strains from the former USSR. The strains were classifiable within subgenotypes A2, D1, D2 and D3. Comparison of the deduced gene products for the four ORFs of 89 genotype D strains revealed 27 subgenotype-specific residues, and a region spanning residues 58-128 in the spacer region of the P gene could be used to distinguish between D1 and D4. This enabled the allocation to subgenotype of strains with partially sequenced genomes. D2 was dominating, while D3 was found in low frequency in the whole region. D1 was most prevalent in the Middle Asian Republics. Mean inter-subgenotype divergences between D1 and D2, D1 and D3 and D2 and D3 were 2.7, 3.4 and 3.4 %, respectively. The intra-subgenotype divergence was 0.4, 1.1, 1.0 and 1.8 % for A2, D1, D2 and D3, respectively. All D1 and D3 strains encoded subtype ayw2, whereas most D2 strains encoded ayw3. Two D2 strains encoded ayw4. Strains with identical S genes were closely related at the level of complete genomes and formed geographically specific clades with low intraclade divergences, possibly indicating past iatrogenic spread. It is not clear whether the finding of four subgenotypes in the area corresponds to separate introductions of the virus or to previous population migrations into the area. An earlier introduction of D3 compared with D2 was supported by its higher intra-subgenotype divergence, while the lower divergence within D1 is probably due to a more recent emergence.