Multi-laboratory evaluation of ReaScan TBE IgM rapid test, 2016 to 2017.

BackgroundTick-borne encephalitis (TBE) is a potentially severe neurological disease caused by TBE virus (TBEV). In Europe and Asia, TBEV infection has become a growing public health concern and requires fast and specific detection.AimIn this observational study, we evaluated a rapid TBE IgM test, ReaScan TBE, for usage in a clinical laboratory setting.MethodsPatient sera found negative or positive for TBEV by serological and/or molecular methods in diagnostic laboratories of five European countries endemic for TBEV (Estonia, Finland, Slovenia, the Netherlands and Sweden) were used to assess the sensitivity and specificity of the test. The patients' diagnoses were based on other commercial or quality assured in-house assays, i.e. each laboratory's conventional routine methods. For specificity analysis, serum samples from patients with infections known to cause problems in serology were employed. These samples tested positive for e.g. Epstein-Barr virus, cytomegalovirus and Anaplasma phagocytophilum, or for flaviviruses other than TBEV, i.e. dengue, Japanese encephalitis, West Nile and Zika viruses. Samples from individuals vaccinated against flaviviruses other than TBEV were also included. Altogether, 172 serum samples from patients with acute TBE and 306 TBE IgM negative samples were analysed.ResultsCompared with each laboratory's conventional methods, the tested assay had similar sensitivity and specificity (99.4% and 97.7%, respectively). Samples containing potentially interfering antibodies did not cause specificity problems.ConclusionRegarding diagnosis of acute TBEV infections, ReaScan TBE offers rapid and convenient complementary IgM detection. If used as a stand-alone, it can provide preliminary results in a laboratory or point of care setting.

Rate of evolution and molecular epidemiology of tick-borne encephalitis virus in Europe, including two isolations from the same focus 44 years apart.

Tick-borne encephalitis virus (TBEV) is a member of the family Flaviviridae. It is transmitted by Ixodes spp. ticks in a cycle involving rodents and small mammals. TBEV has three subtypes: European, Siberian and Far Eastern. The virus causes thousands of cases of meningoencephalitis in Europe annually, with an increasing trend. The increase may be attributed to a complex network of elements, including climatic, environmental and socio-economic factors. In an attempt to understand the evolutionary history and dispersal of TBEV, to existing genetic data we add two novel complete ORF sequences of TBEV strains from northern Europe and the completion of the genome of four others. Moreover, we provide a unique measure for the natural rate of evolution of TBEV by studying two isolations from the same forest on an island in Åland archipelago 44 years apart. For all isolates, we analysed the phylogeny, rate of evolution and probable time of radiation of the different TBEV strains. The results show that the two lineages of TBEV in different Ixodes species have evolved independently for approximately 3300 years. Notably, rapid radiation of TBEV-Eur occurred approximately 300 years ago, without the large-scale geographical clustering observed previously for the Siberian subtype. The measurements from the natural rate of evolution correlated with the estimates done by phylogenetic programs, demonstrating their robustness.

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.

Ixodes persulcatus/pavlovskyi natural hybrids in Siberia: Occurrence in sympatric areas and infection by a wide range of tick-transmitted agents.

Ixodes persulcatus and Ixodes pavlovskyi ticks, two closely related species of the I. ricinus - I. persulcatus group, are widely distributed in the southern part of Western Siberia. Recently, the existence of natural hybrids of I. persulcatus and I. pavlovskyi ticks has been demonstrated. The aim of this study was to evaluate the abundance of I. persulcatus/pavlovskyi hybrids in several locations with different ratios of parental tick species and to investigate the prevalence and genetic variability of a wide range of infectious agents in these hybrids compared to the parental tick species. Natural hybrids of I. persulcatus and I. pavlovskyi ticks were identified in all examined locations in Altai and Novosibirsk, Western Siberia, Russia. The abundance of hybrids varied from 7% to 40% in different locations and was maximal in a location with similar proportions of I. persulcatus and I. pavlovskyi ticks. For the first time, it was shown that hybrids can be infected with the same agents as their parental tick species: tick-borne encephalitis and Kemerovo viruses, Borrelia afzelii, Borrelia bavariensis, Borrelia garinii, Borrelia miyamotoi, Rickettsia helvetica, Rickettsia raoultii, Rickettsia sibirica, "Candidatus Rickettsia tarasevichiae", Anaplasma phagocytophilum, Ehrlichia muris, "Candidatus Neoehrlichia mikurensis", and Babesia microti. The prevalence of most bacterial agents in hybrids was intermediate compared to their parental tick species. Most genetic variants of the identified agents have been previously found in the parental tick species. Wide distribution of I. persulcatus/pavlovskyi natural hybrids implies that I. persulcatus, I. pavlovskyi and their hybrids coexist in all I. persulcatus - I. pavlovskyi sympatric areas.

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.

Multilocus sequence analysis of Borrelia burgdorferi sensu lato isolates from Western Siberia, Russia and Northern Mongolia.

Lyme borreliosis (LB) is the most frequently recorded tick-transmitted disease in Eurasia. Tomsk Province, Western Siberia in Russia and Selenge Aimag in Northern Mongolia are leading regions in the LB incidence rate in these countries. Spirochaetes of the Borrelia burgdorferi sensu lato (s.l.) complex isolated from Ixodes ticks from Tomsk Province (n = 56) and Ixodes persulcatus ticks from Selenge Aimag (n = 5) were genetically characterized using Multi Locus Sequence Typing (MLST), analysis of the 5S23S rRNA intergenic spacer (IGS) amplicons, and p83/100 gene sequencing. According to MLST, B. afzelii (n = 26), B. bavariensis (n = 23), B. garinii (n = 11), and B. valaisiana (n = 1) isolates were detected in Tomsk Province, while B. afzelii and B. bavariensis isolates were identified in Selenge Aimag. Of the 32 revealed sequence types (ST), 21 STs were new and 14 of the new STs belonged to B. afzelii. Several STs of B. afzelii, B. garinii and B. valaisiana identified in this study clustered with European STs found in I. ricinus ticks. Analysis of the 5S23S IGS demonstrated that the studied Borrelia strains showed RFLP pattern characteristic for the following 5S23S IGS types: VS461 (B. afselii), NT29 (B. bavariensis), 20047 (B. bavariensis and B. garinii), VS116 (B. valaisiana), and three new groups (B. afzelii and B. bavariensis). Notably, this is the first report of Asian B. bavariensis possessing a 5S23S IGS RFLP pattern identical to 20047, and analysis of the 5S23S IGS did not provide correct determination of Borrelia species occurring in Asia. Genotyping of Borrelia strains using the clpA, pepX, and p83/100 genes demonstrated the same result as genotyping based on MLST; and further investigations are required to confirm that these three genetic loci could be used for determination of bacterial species from the B. burgdorferi s.l. complex because data based on single loci may be misleading.

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.

Rapid subtyping of tick-borne encephalitis virus isolates using multiplex RT-PCR.

Tick-borne encephalitis virus, an emerging pathogen in several countries in Europe and Asia, has been divided into three subtypes (European, Siberian and Far Eastern). These subtypes are associated with different severities of the disease. For that reason, early determination of the subtype in a clinical sample or in ticks removed from a patient in areas of co-circulation of two or three subtypes is of high importance. The development of a simple method of multiplex RT-PCR for rapid and easy subtyping of tick-borne encephalitis virus isolates is reported to fill this requirement. The method is based on the unique combination of oligonucleotide primers hybridizing with subtype-specific "signature" positions of the sequence encoding the viral envelope protein. The developed multiplex RT-PCR also appears to be a useful method in studies focused on the molecular-epidemiology of tick-borne encephalitis virus.

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.

Prevalence of Antibodies Against Hepatitis E Virus in Veterinarians in Estonia.

In this cross-sectional study, we investigated veterinarians in Estonia for evidence of exposure to hepatitis E virus (HEV). In 2012, we collected sera from 158 persons attending a veterinary conference, of whom 156 completed a questionnaire covering their background information. Altogether 115 persons reported they had obtained a veterinary degree and were included in this study. The sera were tested for presence of antibodies against HEV using a commercial enzyme linked immunosorbent assay and a commercial immunoblot assay in series. A sample was considered antibody-positive if it tested positive with both tests. Antibody-positive samples were further examined for the presence of HEV RNA. Three (2.6%) of the 115 veterinarians tested positive for immunoglobulin G antibodies against HEV, whereas no immunoglobulin M antibodies against the virus were detected. The antibody-positive veterinarians were small animal practitioners. Pigs comprised no or small part of their working time or patients. No HEV RNA was detected in the antibody-positive samples. The prevalence of antibodies against HEV in veterinarians in Estonia was lower than what has been observed in veterinarians in other countries.

New genetic lineage within the Siberian subtype of tick-borne encephalitis virus found in Western Siberia, Russia.

Tick-borne encephalitis virus (TBEV), a member of the Flaviviridae family, is a causative agent of a severe neurological disease. There are three main TBEV subtypes: the European (TBEV-Eu), Far Eastern (TBEV-FE), and Siberian (TBEV-Sib). Currently, three lineages within TBEV-Sib have been recorded. In this study, the genetic and biological characteristics of a new original strain, TBEV-2871, isolated in the Novosibirsk province of Western Siberia, Russia were investigated. The strain has low neuroinvasiveness in mice. Phylogenetic analysis demonstrated that TBEV-2871 belongs to TBEV-Sib, but does not cluster with any of the TBEV-Sib lineages. The TBEV-2871 strain has 88-89% nucleotide sequence identity with the other TBEV-Sib strains, 84-86% nucleotide sequence identity with the TBEV-FE and TBEV-Eu subtypes and is genetically close to the subtype division border. The TBEV-2871 polyprotein sequence includes 43 unique amino acid substitutions, 30 of which are recorded at positions that are conserved among all TBEV subtypes. Strain TBEV-2871 and two similar but not identical isolates found in Kemerovo province, Western Siberia are separated into a new lineage tentatively named Obskaya after the name of Ob riber, in the vicinity of which the TBEV-2871 was first found. A molecular evolution investigation demonstrated that within TBEV-Sib, the Obskaya lineage likely separated 1535years ago, which is even earlier than the Baltic lineage.

Serological Evidence of Exposure to Globally Relevant Zoonotic Parasites in the Estonian Population.

We investigated Estonian population and its selected subgroups for serological evidence of exposure to Ascaris lumbricoides, Echinococcus spp., Taenia solium, Toxocara canis, Toxoplasma gondii, and Trichinella spiralis. Serum samples from 999 adults representing general population, 248 children aged 14-18, 158 veterinarians, 375 animal caretakers, and 144 hunters were tested for specific immunoglobulin G antibodies against the selected parasites using commercial enzyme immunoassays (ELISA). Sera yielding positive or twice grey zone Echinococcus spp, T. solium, T. canis, and T. spiralis results were subjected to western blot (WB) analysis. In the general population, based on the ELISA results, the A. lumbricoides seroprevalence was 12.7%, Echinococcus spp. seroprevalence was 3.3%, T. solium seroprevalence was 0.7%, T. canis seroprevalence was 12.1%, T. gondii seroprevalence was 55.8%, and T. spiralis seroprevalence was 3.1%. Ascaris lumbricoides seroprevalences were higher in children and in animal caretakers than in the general population, and T. canis seroprevalence was higher in animal caretakers than in the general population. Compared with the general population, Echinococcus spp. seroprevalence was higher in children. By contrast, T. gondii seroprevalence was higher in animal caretakers, and lower in children, than in the general population. In the general population, the WB-confirmed Echinococcus spp. seroprevalence was 0.5%, T. solium cysticercosis seroprevalence was 0.0%, Toxocara spp. seroprevalence was 14.5%, and Trichinella spp. seroprevalence was 2.7%. WB-confirmed Toxocara spp. seroprevalence was higher in animal caretakers than in the general population. We found serological evidence of exposure to zoonotic parasites in all tested groups. This calls for higher awareness of zoonotic parasitic infections in Estonia.

First evidence of established populations of the taiga tick Ixodes persulcatus (Acari: Ixodidae) in Sweden.

BACKGROUND: The tick species Ixodes ricinus and I. persulcatus are of exceptional medical importance in the western and eastern parts, respectively, of the Palaearctic region. In Russia and Finland the range of I. persulcatus has recently increased. In Finland the first records of I. persulcatus are from 2004. The apparent expansion of its range in Finland prompted us to investigate if I. persulcatus also occurs in Sweden. METHODS: Dog owners and hunters in the coastal areas of northern Sweden provided information about localities where ticks could be present. In May-August 2015 we used the cloth-dragging method in 36 localities potentially harbouring ticks in the Bothnian Bay area, province Norrbotten (NB) of northern Sweden. Further to the south in the provinces Västerbotten (VB) and Uppland (UP) eight localities were similarly investigated. RESULTS: Ixodes persulcatus was detected in 9 of 36 field localities in the Bothnian Bay area. Nymphs, adult males and adult females (n = 46 ticks) of I. persulcatus were present mainly in Alnus incana - Sorbus aucuparia - Picea abies - Pinus sylvestris vegetation communities on islands in the Bothnian Bay. Some of these I. persulcatus populations seem to be the most northerly populations so far recorded of this species. Dog owners asserted that their dogs became tick-infested on these islands for the first time 7-8 years ago. Moose (Alces alces), hares (Lepus timidus), domestic dogs (Canis lupus familiaris) and ground-feeding birds are the most likely carriers dispersing I. persulcatus in this area. All ticks (n = 124) from the more southern provinces of VB and UP were identified as I. ricinus. CONCLUSIONS: The geographical range of the taiga tick has recently expanded into northern Sweden. Increased information about prophylactic, anti-tick measures should be directed to people living in or visiting the coastal areas and islands of the Baltic Bay.

Genetic Spectrum of Idiopathic Restrictive Cardiomyopathy Uncovered by Next-Generation Sequencing.

BACKGROUND: Cardiomyopathies represent a rare group of disorders often of genetic origin. While approximately 50% of genetic causes are known for other types of cardiomyopathies, the genetic spectrum of restrictive cardiomyopathy (RCM) is largely unknown. The aim of the present study was to identify the genetic background of idiopathic RCM and to compile the obtained genetic variants to the novel signalling pathways using in silico protein network analysis. PATIENTS AND METHODS: We used Illumina MiSeq setup to screen for 108 cardiomyopathy and arrhythmia-associated genes in 24 patients with idiopathic RCM. Pathogenicity of genetic variants was classified according to American College of Medical Genetics and Genomics classification. RESULTS: Pathogenic and likely-pathogenic variants were detected in 13 of 24 patients resulting in an overall genotype-positive rate of 54%. Half of the genotype-positive patients carried a combination of pathogenic, likely-pathogenic variants and variants of unknown significance. The most frequent combination included mutations in sarcomeric and cytoskeletal genes (38%). A bioinformatics approach underlined the mechanotransducing protein networks important for RCM pathogenesis. CONCLUSIONS: Multiple gene mutations were detected in half of the RCM cases, with a combination of sarcomeric and cytoskeletal gene mutations being the most common. Mutations of genes encoding sarcomeric, cytoskeletal, and Z-line-associated proteins appear to have a predominant role in the development of RCM.

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.

Prevalence of tick-borne encephalitis virus in Ixodes ricinus ticks in northern Europe with particular reference to Southern Sweden.

BACKGROUND: In northern Europe, the tick-borne encephalitis virus (TBEV) of the European subtype is usually transmitted to humans by the common tick Ixodes ricinus. The aims of the present study are (i) to obtain up-to-date information on the TBEV prevalence in host-seeking I. ricinus in southern and central Sweden; (ii) to compile and review all relevant published records on the prevalence of TBEV in ticks in northern Europe; and (iii) to analyse and try to explain how the TBE virus can be maintained in natural foci despite an apparently low TBEV infection prevalence in the vector population. METHODS: To estimate the mean minimum infection rate (MIR) of TBEV in I. ricinus in northern Europe (i.e. Denmark, Norway, Sweden and Finland) we reviewed all published TBEV prevalence data for host-seeking I. ricinus collected during 1958-2011. Moreover, we collected 2,074 nymphs and 906 adults of I. ricinus from 29 localities in Sweden during 2008. These ticks were screened for TBEV by RT-PCR. RESULTS: The MIR for TBEV in nymphal and adult I. ricinus was 0.28% for northern Europe and 0.23% for southern Sweden. The infection prevalence of TBEV was significantly lower in nymphs (0.10%) than in adult ticks (0.55%). At a well-known TBEV-endemic locality, Torö island south-east of Stockholm, the TBEV prevalence (MIR) was 0.51% in nymphs and 4.48% in adults of I. ricinus. CONCLUSIONS: If the ratio of nymphs to adult ticks in the TBEV-analysed sample differs from that in the I. ricinus population in the field, the MIR obtained will not necessarily reflect the TBEV prevalence in the field. The relatively low TBEV prevalence in the potential vector population recorded in most studies may partly be due to: (i) inclusion of uninfected ticks from the 'uninfected areas' surrounding the TBEV endemic foci; (ii) inclusion of an unrepresentative, too large proportion of immature ticks, compared to adult ticks, in the analysed tick pools; and (iii) shortcomings in the laboratory techniques used to detect the virus that may be present in a very low concentration or undetectable state in ticks which have not recently fed.

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.