Norovirus strains among children aged 0-18 years hospitalized with acute gastroenteritis in Estonia 2015-2016.

Norovirus (NoV) is the leading cause of acute gastroenteritis (AGE) in many countries that have introduced universal rotavirus mass vaccination. This is the first study to report data on NoV strains in Estonia. We recruited 2249 children aged 0-18 years hospitalized for AGE in Estonian hospitals from February 1, 2015 to August 31, 2016. Norovirus gastroenteritis (NoVGE) was diagnosed in 14.5% (n = 325) cases. Stool sample for RNA extraction and genotyping was available in 86% (n = 280) of NoVGE cases (2015, n = 91; 2016, n = 189). Dominant capsid types detected in 75% (n = 210) samples were, GII.4 (63.8%, n = 134), GII.3 (15.2%, n = 32), GII.17 (6.7%, n = 14), and GII.6 (5.2%, n = 11). Prevailing RNA polymerase types found in 77% (n = 215) samples were GII.P31 (51.1%, n = 110), GII.P21 (17.7%, n = 38), GII.P4 (11.2%, n = 24), and GII.P7 (6.5%, n = 14). Both regions were typeable for 67% (n = 189) of samples. Most prevalent strains were GII.4Sydney_2012[P31] (48.7%, n = 92), GII.3[P21] (15.3%, n = 29), GII.4Sydney_2012[P4] (5.8%, n = 11) and GII.17[P17] (5.8%, n = 11). Simpson's diversity index showed a significant difference between the age groups 1-4 and 5-9 years: D 0.64 (95% confidence interval [CI]: 0.55-0.73) versus 0.83 (95% CI: 0.81-0.86), respectively (p = 0.03). An accurate understanding of NoV strain diversity is important for control and preventive measures, especially in the postrotavirus vaccine era.

Evaluation of Prevalence of Hepatitis E Clinical Markers among Donors in Estonia.

Hepatitis E virus (HEV) is now considered the most common cause of acute hepatitis worldwide. There are no published data about the prevalence of antibodies to HEV and RNA in donor sera in Estonia, and this precludes planning measures for preventing HEV proliferation through blood transfusion services. Here, were report data from an analysis of 1002 sera on the prevalence of anti-HEV IgG and IgM and the viral RNA. The antibodies were found in 48 donor sera (4.8%); of these, 40 (4%) harbored anti-HEV IgG, 15 (1.5%) contained anti-HEV IgM, and 7 donors had anti-HEV antibodies of both classes simultaneously. HEV RNA was not detected in any blood serum. Statistical associations of infection risk factors (gender, age, travel in the last six months, contact with pigs and/or wild boars in the last six months, consumption of thermally unprocessed/raw pork or boar meat, raw/unfiltered tap water or water from natural sources, unpasteurized farm dairy products, and unwashed berries and/or vegetables) were assessed. None of the listed factors were found to be associated with a higher or lower risk of anti-HEV antibody presence. At the same time, an increasing share of anti-HEV IgG carriers with age was found. The absence of HEV RNA in the analyzed donor plasma samples proves that HEV acute infection prevalence in Estonia does not exceed the average level of European countries. There is no urgent necessity to enter a requirement for a total screening of blood plasma for HEV RNA prevalence in Estonia.

First Expert Elicitation of Knowledge on Possible Drivers of Observed Increasing Human Cases of Tick-Borne Encephalitis in Europe.

Tick-borne encephalitis (TBE) is a viral disease endemic in Eurasia. The virus is mainly transmitted to humans via ticks and occasionally via the consumption of unpasteurized milk products. The European Centre for Disease Prevention and Control reported an increase in TBE incidence over the past years in Europe as well as the emergence of the disease in new areas. To better understand this phenomenon, we investigated the drivers of TBE emergence and increase in incidence in humans through an expert knowledge elicitation. We listed 59 possible drivers grouped in eight domains and elicited forty European experts to: (i) allocate a score per driver, (ii) weight this score within each domain, and (iii) weight the different domains and attribute an uncertainty level per domain. An overall weighted score per driver was calculated, and drivers with comparable scores were grouped into three terminal nodes using a regression tree analysis. The drivers with the highest scores were: (i) changes in human behavior/activities; (ii) changes in eating habits or consumer demand; (iii) changes in the landscape; (iv) influence of humidity on the survival and transmission of the pathogen; (v) difficulty to control reservoir(s) and/or vector(s); (vi) influence of temperature on virus survival and transmission; (vii) number of wildlife compartments/groups acting as reservoirs or amplifying hosts; (viii) increase of autochthonous wild mammals; and (ix) number of tick species vectors and their distribution. Our results support researchers in prioritizing studies targeting the most relevant drivers of emergence and increasing TBE incidence.

Rickettsia spp. in rodent-attached ticks in Estonia and first evidence of spotted fever group Rickettsia species Candidatus Rickettsia uralica in Europe.

BACKGROUND: Rickettsia spp. are human pathogens that cause a number of diseases and are transmitted by arthropods, such as ixodid ticks. Estonia is one of few regions where the distribution area of two medically important tick species, Ixodes persulcatus and I. ricinus, overlaps. The nidicolous rodent-associated Ixodes trianguliceps has also recently been shown to be present in Estonia. Although no data are available on human disease(s) caused by tick-borne Rickettsia spp. in Estonia, the presence of three Rickettsia species in non-nidicolous ticks has been previously reported. The aim of this study was to detect, identify and partially characterize Rickettsia species in nidicolous and non-nidicolous ticks attached to rodents in Estonia. RESULTS: Larvae and nymphs of I. ricinus (n = 1004), I. persulcatus (n = 75) and I. trianguliceps (n = 117), all removed from rodents and shrews caught in different parts of Estonia, were studied for the presence of Rickettsia spp. by nested PCR. Ticks were collected from 314 small animals of five species [Myodes glareolus (bank voles), Apodemus flavicollis (yellow necked mice), A. agrarius (striped field mice), Microtus subterranius (pine voles) and Sorex araneus (common shrews)]. Rickettsial DNA was detected in 8.7% (103/1186) of the studied ticks. In addition to identifying R. helvetica, which had been previously found in questing ticks, we report here the first time that the recently described I. trianguliceps-associated Candidatus Rickettsia uralica has been identified west of the Ural Mountains.

Association of the Sialylation of Antibodies Specific to the HCV E2 Envelope Glycoprotein with Hepatic Fibrosis Progression and Antiviral Therapy Efficacy.

The E2 envelope glycoprotein of the hepatitis C virus (HCV) is a major target of broadly neutralizing antibodies that are closely related to a spontaneous cure of HCV infection. There is still no data about the diversity of E2-specific antibodies (Abs) glycosylation. The aim of this study was to analyze the level and sialylation of E2 IgG Abs, the relation of the respective changes to hepatic fibrosis (F) progression and their possible association with the efficacy of interferon-α-2a plus ribavirin (IFN-RBV) antiviral therapy. One hundred three HCV infected treatment-naive patients were examined using ELISA with E2 recombinant protein as antigen and sialic acid-specific Sambucus nigra agglutinin. The efficacy of the IFN-RBV treatment of patients with HCV dominant 1b and 3a genotypes (GT) was evaluated. A significant decrease of E2 Abs sialylation in the late stages of fibrosis was found irrespective of HCV genotype. On this basis, the F4 stage of fibrosis can be discriminated from its F0 or F1-3 stage by an about 75-79% accuracy. HCV infection of 1b genotype is associated with the production of lower sialylated E2 Abs, a higher frequency of F4 stage fibrosis, and a worse response to antiviral therapy. The increased SNA reactivity of E2 Abs was observed in patients with a sustained virological response (SVR). The proportion of SVR responders was significantly higher among patients with 3a genotype. However, for both dominant HCV genotypes (3a and 1b), an increased sialylation of E2 IgG was associated with a higher rate of patients with sustained virological response to antiviral therapy. Thus, the association of alterations of anti-E2 IgG Abs sialylation with hepatic fibrosis stage, HCV genotype, and the efficacy of antiviral therapy enables using these changes as novel noninvasive predictive biomarkers. The clinical potential of these findings is discussed.

IgG Antibodies to GlcNAcß and Asialo-GM2 (GA2) Glycans as Potential Markers of Liver Damage in Chronic Hepatitis C and the Efficacy of Antiviral Treatment.

Total serum IgG level is a surrogate marker of hepatitis C (HC) severity. Antibodies (Abs) to microbial glycans could be markers of HC severity caused by the translocation of microbial products. The level of anti-glycan (AG) Abs was analysed in serum samples of patients (n = 128) with chronic HC in ELISA using fourteen synthetic glycans present in microbes and adhesins to evaluate the association of Abs with clinical parameters and the efficacy of antiviral treatment. The anti-GlcNAcß IgG level was significantly higher in patients with fibrosis (P = 0.021) and severe portal inflammation (P < 0.001) regardless of other clinical parameters. The ROC curve analysis showed sensitivity of 0.59, specificity of 0.84, and AUC of 0.71 in discriminating F0 from F1-4 (HCV genotype-1b-infected patients). The level of anti-GA2 Abs before Peg-IFN/RBV treatment was significantly higher in nonsustained viral response (non-SVR) to treatment than in SVR (P = 0.033). ROC analysis showed sensitivity of 0.62, specificity of 0.70, and AUC of 64. Correlations of AG Abs to clinical parameters were found. The quantification of anti-GlcNAcß Abs deserves attention in assessment of the hepatic damage while anti-GA2 Abs may be a sign of immune response related to the antiviral treatment.

Hepatitis E virus infection in different groups of Estonian patients and people who inject drugs.

BACKGROUND: Previously we demonstrated a high prevalence of hepatitis E virus (HEV) in domestic pigs and wild boars, the main reservoir and possible source of HEV infections in humans. But so far there are no reports about spread of HEV in Estonian human population. OBJECTIVES: The present study aimed to determine the prevalence and genotyping of HEV in different groups of the Estonian adult population. STUDY DESIGN: Totally 1426 human serum samples were tested (763 patients with clinically diagnosed nonA/B/C hepatitis, 176 hemodialysis patients, 282 patients with suspected HEV infection and 205 people who injected drugs (PWID)). Presence of anti-HEVantibodies was assessed by ELISA and confirmed by immunoblotting. All anti-HEV positive sera were analyzed for RNA by qPCR. Amplified ORF2 region was sequenced and used for phylogenetic analysis. RESULTS: Antibody assay revealed 49 samples from 1426 (3.4%) with acute (17) or past (32) HEV infection. HEV RNA was detected in 10 anti-HEV IgM positive samples, including 9 samples from patients with suspected HEV infection and 1 hemodialysis patient. Anti-HEV IgG were found in 7.8% patients with suspected HEV infection, in 4% hemodialysis patients, in 2.4% PWID and in 1.96% patients with nonA/B/C hepatitis. All groups demonstrated a trend to share of anti-HEV seroprevalence increasing with age. Phylogenetic analysis of 9 HEV RNA sequences revealed that 3 sequences belonged to HEV genotype 1; 6 ones to genotype 3 (1 sequence belonged to sub-genotype 3a, two ones - sub-genotype 3e, and three ones - to sub-genotype 3f). CONCLUSIONS: Despite the high seroprevalence among domestic pigs, no evidence of HEV transmission from Estonian pigs to humans was found. The results of our study suggest that HEV infections in Estonia are most likely associated with travel or with consumption of imported food products.

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.

Identification of I. ricinus, I. persulcatus and I. trianguliceps species by multiplex PCR.

Correct identification of tick species is an essential requirement for any scientific study engaged in tick-associated research. However, morphological identification can lead to misinterpretations, especially when dealing with vector-host research and sub-adult, engorged or damaged specimens. To overcome this limitation, we developed a novel assay to discriminate between Ixodes ricinus, I. persulcatus and I. trianguliceps species collected from rodents or vegetation, using the second internal transcribed spacer (ITS2) as a genetic marker. This single tube multiplex PCR allows specific amplification of targeted species and produces rapid and accurate results. The specificity was confirmed by sequencing the ITS2 and partial 16S rRNA genes from ticks collected from Estonia, Latvia, Sweden and Russia. We tested the assay in a large-scale experiment, and a total of 1284 ticks removed from rodents and shrews were successfully identified at species level.

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.

Hepatitis E Virus in Domestic Pigs, Wild Boars, Pig Farm Workers, and Hunters in Estonia.

While hepatitis E is a growing health concern in Europe, epidemiological data on hepatitis E virus (HEV) in Estonia are scarce. Along with imported HEV infections, autochthonous cases are reported from European countries. Both domestic and wild animals can be a source of human cases of this zoonosis. Here, we investigated the presence of anti-HEV antibodies and HEV RNA in domestic pigs and wild boars, as well as in pig farm workers and hunters in Estonia. Anti-HEV antibodies were detected in 234/380 (61.6%) of sera from domestic pigs and in all investigated herds, and in 81/471 (17.2%) of meat juice samples from wild boars. HEV RNA was detected by real-time PCR in 103/449 (22.9%) of fecal samples from younger domestic pigs and 13/81 (16.0%) of anti-HEV-positive wild boar samples. Analysis of sera from 67 pig farm workers and 144 hunters revealed the presence of HEV-specific IgG in 13.4 and 4.2% of the samples, respectively. No HEV RNA was detected in the human serum samples. Phylogenetic analyses of HEV sequences from domestic pigs and wild boars, based on a 245 bp fragment from the open reading frame 2 showed that all of them belonged to genotype 3. The present study demonstrates the presence of HEV in Estonian domestic pig and wild boar populations, as well as in humans who have direct regular contact with these animals. Our results suggest that HEV infections are present in Estonia and require attention.

Genetic characterization of the human relapsing fever spirochete Borrelia miyamotoi in vectors and animal reservoirs of Lyme disease spirochetes in France.

BACKGROUND: In France as elsewhere in Europe the most prevalent TBD in humans is Lyme borreliosis, caused by different bacterial species belonging to Borrelia burgdorferi sensu lato complex and transmitted by the most important tick species in France, Ixodes ricinus. However, the diagnosis of Lyme disease is not always confirmed and unexplained syndromes occurring after tick bites have become an important issue. Recently, B. miyamotoi belonging to the relapsing fever group and transmitted by the same Ixodes species has been involved in human disease in Russia, the USA and the Netherlands. In the present study, we investigate the presence of B. miyamotoi along with other Lyme Borreliosis spirochetes, in ticks and possible animal reservoirs collected in France. METHODS: We analyzed 268 ticks (Ixodes ricinus) and 72 bank voles (Myodes glareolus) collected and trapped in France for the presence of DNA from B. miyamotoi as well as from Lyme spirochetes using q-PCR and specific primers and probes. We then compared the French genotypes with those found in other European countries. RESULTS: We found that 3% of ticks and 5.55% of bank voles were found infected by the same B. miyamotoi genotype, while co-infection with other Lyme spirochetes (B. garinii) was identified in 12% of B. miyamotoi infected ticks. Sequencing showed that ticks and rodents carried the same genotype as those recently characterized in a sick person in the Netherlands. CONCLUSIONS: The genotype of B. miyamotoi circulating in ticks and bank voles in France is identical to those already described in ticks from Western Europe and to the genotype isolated from a sick person in The Netherlands. This results suggests that even though no human cases have been reported in France, surveillance has to be improved. Moreover, we showed that ticks could simultaneously carry B. miyamotoi and Lyme disease spirochetes, increasing the problem of co-infection in humans.

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.

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 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.

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.