Phylogenetic characterisation of tick-borne encephalitis virus from Lithuania.

The Baltic states are the region in Europe where tick-borne encephalitis (TBE) is most endemic. The highest notification rate of TBE cases is reported in Lithuania, where the incidence of TBE has significantly increased since 1992. A recent study reported 0.4% prevalence of TBE virus (TBEV) in the two most common tick species distributed in Lithuania, Ixodes ricinus and Dermacentor reticulatus, with the existence of endemic foci confirmed in seven out of Lithuania's ten counties. However, until now, no comprehensive data on molecular characterisation and phylogenetic analysis have been available for the circulating TBEV strains. The aim of this study was to analyse TBEV strains derived from I. ricinus and D. reticulatus ticks collected from Lithuania and provide a genotypic characterisation of viruses based on sequence analysis of partial E protein and NS3 genes. The 54 nucleotide sequences obtained were compared with 81 TBEV strains selected from the NCBI database. Phylogenetic analysis of the partial E and NS3 gene sequences derived from 34 Lithuanian TBEV isolates revealed that these were specific to Lithuania, and all belonged to the European subtype, with a maximum identity to the Neudoerfl reference strain (GenBank accession no. U27495) of 98.7% and 97.4%, respectively. The TBEV strains showed significant regional genetic diversity. The detected TBEV genotypes were not specific to the tick species. However, genetic differences were observed between strains from different locations, while strains from the same location showed a high similarity.

Molecular detection of Babesia vesperuginis in bats from Lithuania.

Babesia vesperuginis is an intraerythrocytic protozoan parasite that circulates among bats and ticks in many countries worldwide. However, the distribution of B. vesperuginis in the Baltic region has not been studied. A total of 86 dead bats from eight different species were collected and screened for Babesia spp. using real-time PCR. Overall, 52.3% (45/86) of the bats were found positive for Babesia spp. The prevalence of Babesia spp. in different organs varied, with the highest prevalence observed in heart tissues (37.0%) and the lowest in liver tissues (22.2%). However, the observed differences in prevalence among organs were not statistically significant. Blood samples from 125 bats of nine different species were also analyzed for Babesia spp. prevalence using real-time PCR and nested PCR. The results showed a prevalence of 35.2% and 22.4%, respectively. Moreover, 28.3% (17/60) of the examined blood samples were confirmed positive for Babesia spp. through blood smear analysis. The total of 32 partial sequences of the 18S rRNA gene derived in this study were 100% identical to B. vesperuginis sequences from GenBank. In eight species of bats, Pipistrellus nathusii, Pipistrellus pipistrellus, Pipistrellus pygmaeus, Vespertilio murinus, Eptesicus nilssonii, Eptesicus serotinus, Myotis daubentonii and Nyctalus noctula, Babesia parasites were identified. In E. nilssonii, Babesia spp. was identified for the first time.

Molecular Identification and Phylogenetic Analysis of Laelapidae Mites (Acari: Mesostigmata).

The family Laelapidae (Dermanyssoidea) is morphologically and ecologically the most diverse group of Mesostigmata mites. Although molecular genetic data are widely used in taxonomic identification and phylogenetic analysis, most classifications in Mesostigmata mites are based solely on morphological characteristics. In the present study, eight species of mites from the Laelapidae (Dermanyssoidea) family collected from different species of small rodents in Lithuania, Norway, Slovakia, and the Czech Republic were molecularly characterized using the nuclear (28S ribosomal RNA) and mitochondrial (cytochrome oxidase subunit I gene) markers. Obtained molecular data from 113 specimens of mites were used to discriminate between species and investigate the phylogenetic relationships and genetic diversity among Laelapidae mites from six genera. This study provides new molecular data on Laelaps agilis, Laelaps hilaris, Laelaps jettmari, Haemogamasus nidi, Eulaelaps stabularis, Hyperlaelaps microti, Myonyssus gigas, and Hirstionyssus sp. mites collected from different rodent hosts and geographical regions in Europe.

Genetic Diversity of Babesia canis Strains in Dogs in Lithuania.

Canine babesiosis is an emerging and rapidly expanding tick-borne disease in central and northeast Europe. In the last two decades, the endemic area of Babesia canis has expanded from central Europe to the Baltic region. This study aimed to investigate the genetic diversity of B. canis strains isolated from naturally infected dogs in different regions of Lithuania using PCR-RFLP and sequence analyses based on a partial region of 18S rRNA and Bc28.1 genes. Blood samples from 149 dogs suspected of having babesiosis were collected in Lithuania during 2016-2017. Based on PCR-RFLP profiles and two nucleotide substitutions observed in 18S rRNA gene sequences, three B. canis genotypes were identified in Lithuania-18S rRNA-A, 18S rRNA-B and 18S rRNA-A/B-with the A/B genotype predominating (83.9%). Based on the obtained PCR-RFLP profiles of the Bc28.1 gene, four B. canis genotypes were identified: Bc28.1-B (53.8%), Bc28.1-34 (20.8%), Bc28.1-A (17.9%), and Bc28.1-34/A or B (7.5%). Sequence analysis of the partial Bc28.1 gene revealed eighteen polymorphic sites and thirteen sequence variants among the Lithuanian samples. The B. canis genotypes obtained were detected with varying prevalences in different regions of Lithuania.

Babesia microti in Rodents from Different Habitats of Lithuania.

Babesia microti (Aconoidasida: Piroplasmida) (Franca, 1910) is an emerging tick-borne parasite with rodents serving as the considered reservoir host. However, the distribution of B. microti in Europe is insufficiently characterized. Based on the sample of 1180 rodents from 19 study sites in Lithuania, the objectives of this study were: (1) to investigate the presence of Babesia parasites in eight species of rodents, (2) to determine the prevalence of Babesia parasites in rodents from different habitats, and (3) to characterize the detected Babesia strains using partial sequencing of the 18S rRNR gene. Babesia DNA was detected in 2.8% rodents. The highest prevalence of Babesia was found in Microtus oeconomus (14.5%) and Microtus agrestis (7.1%) followed by Clethrionomys glareolus (2.3%), Apodemus flavicollis (2.2%) and Micromys minutus (1.3%). In M.minutus, Babesia was identified for the first time. The prevalence of Babesia-infected rodents was higher in the meadow (5.67%) than in the ecotone (1.69%) and forest (0.31%) habitats. The sequence analysis of the partial 18S rRNA gene reveals that Babesia isolates derived from rodents were 99-100% identical to human pathogenic B. microti 'Jena/Germany' strain.

Prevalence and diversity of Bartonella species in small rodents from coastal and continental areas.

Worldwide, Bartonella infections are known to inflict a wide range of mammals and, within rodents alone, more than 20 Bartonella species have been detected. There is, however, a lack of studies on the presence of Bartonella spp. in rodents in the Baltic region. We analysed 580 individuals belonging to eight small rodent species trapped in coastal and continental areas of Lithuania during 2015-2016. The presence of Bartonella DNA was examined by real-time PCR targeting the ssrA gene. The molecular characterization of the bacteria strains was based on sequence analysis of two housekeeping genes (rpoB, groEL) and the intergenic spacer region (ITS). For the rodents overall, the prevalence of Bartonella spp. was 54.8%, while the prevalence figures for each of the individual species were 8.3% in M. musculus, 15.8% in A. agrarius, 33.3% in M. arvalis, 42.4% in M. glareolus, 53.4% in M. oeconomus, 57.5% in M. minutus, 79.6% in A. flavicollis to 80% in M. agrestis. Sequence analysis revealed that the Bartonella strains belonged to the B. grahamii, B. taylorii, B. rochalimae, B. tribocorum, B. coopersplainsensis and B. doshiae genogroups. The highest Bartonella infection rates and the highest species diversity were both detected in rodents captured in the coastal area. To our knowledge, these are the first reports of the presence of B. coopersplainsensis, B. doshiae and B. tribocorum in Lithuania.

Prevalence and diversity of Rickettsia species in ectoparasites collected from small rodents in Lithuania.

BACKGROUND: Rickettsiae are emerging pathogens causing public health problems in many countries around the world. Rickettsia spp. are found in association with a wide range of arthropods which feed on different species of animals. However, the distribution and natural cycle of Rickettsia species and their association with different arthropod vectors are not fully established. The aim of this study was to investigate the presence and prevalence of Rickettsia spp. in ticks, mites and fleas parasitizing different species of small mammals in Lithuania and to molecularly characterize the Rickettsia spp. obtained from different ectoparasites. RESULTS: A total of 1261 ectoparasites (596 Ixodes ricinus ticks, 550 mites of five species and 115 fleas of eight species) collected from 238 rodents in Lithuania during 2013-2014 were investigated for the presence of Rickettsia pathogens. Infection rates were calculated as the maximum likelihood estimation (MLE) with 95% confidence intervals (CI). The infection rate varied among ectoparasites and was found highest in fleas 43.5%, followed by I. ricinus ticks (MLE = 26.5%; 95% CI: 22.2-31.3%) and then mites (MLE = 9.3%; 95% CI: 7.0-12.2%). Sequence analysis of partial gltA and 17kDa genes revealed the presence of Rickettsia helvetica, R. felis, R. monacensis, Rickettsia sp. and rickettsial endosymbionts. Four Rickettsia spp. were identified in fleas, while three Rickettsia spp. were identified in Laelapidae mites and only one (R. helvetica) in I. ricinus ticks. CONCLUSIONS: To our knowledge, this is the first report of the occurrence and molecular characterization of Rickettsia spp. in 11 species of ectoparasites of small rodents in Lithuania. The present data extend the knowledge on the distribution of Rickettsia spp. and their association with different arthropod vectors. Prior to our study, R. felis had never been identified in Lithuania. To our knowledge, this is also the first report of R. felis in L. agilis and H. microti mites and in Ct. agyrtes and H. talpae fleas, as well as the first detection of R. monacensis in Ct. agyrtes fleas.

Investigation of Babesia spp. in sympatric populations of Dermacentor reticulatus and Ixodes ricinus ticks in Lithuania and Latvia.

The objectives of the present study were to investigate the presence of the Babesia parasites in Dermacentor reticulatus ticks along its current distribution range in Lithuania and Latvia, and Ixodes ricinus in Lithuania, to characterize the detected Babesia spp. using partial sequencing of 18S rRNR gene, and to determine the prevalence of the Babesia pathogens in ticks from different locations of Lithuania and Latvia. From 2013 to 2015, four D. reticulatus nymphs and 2255 questing adults were collected from 40 locations in Lithuania, and 181 questing adult D. reticulatus specimens from 11 locations in Latvia. In Lithuania, 16 questing I. ricinus nymphs and 354 questing adults were collected from 12 locations with the sympatric existence of D. reticulatus and I. ricinus. In Lithuania, Babesia spp. were detected in 1.2% (26/2259) of D. reticulatus and in 9.5% (35/370) of I. ricinus ticks. The overall prevalence of Babesia in D. reticulatus ticks from Latvia was 2.8% (5/181). Sequence analysis of partial 18S rRNA gene of positive samples indicated the presence of B. canis and B. venatorum in D. reticulatus ticks, whereas B. microti and B. venatorum were detected in I. ricinus ticks. Our study is the first investigation on the prevalence and molecular characterization of zoonotic B. canis, B. venatorum, and B. microti in ixodid ticks in Lithuania, and of B. canis in D. reticulatus ticks in Latvia. It is also the first report of B. venatorum in D. reticulatus ticks.

New localities of Dermacentor reticulatus ticks in the Baltic countries.

According to previous observations in three Baltic States, Dermacentor reticulatus was found only in Lithuania where it occurred mainly in the central and western parts of the country. During the past decade, evidence about a changing distribution of D. reticulatus in the Baltic countries was provided by the occurrence of canine babesiosis in the new locations in Lithuania and Latvia. In the present study the current distribution of D. reticulatus in Lithuania and Latvia was investigated. Ticks were collected in different habitats in 2013-2014. A total of 3693 questing ticks belonging to D. reticulatus (n = 2789), Ixodes ricinus (n = 896) and I. persulcatus (n=8) were collected in Lithuania and Latvia. Questing D. reticulatus ticks were found in 73.4% (58/79) and in 44.4% (12/28) of the sampling localities in Lithuania and Latvia, respectively. Relative abundances of the ticks in different habitats were compared. All sites with high abundance of D. reticulatus ticks were localized in open areas close to a water basin and mixed forest. The present study demonstrates that during the past two decades D. reticulatus has expanded its range in the Baltic countries. D. reticulatus has been detected in 38 new localities in which this species had not been previously reported. The northern border of D. reticulatus in central Europe moved further to the north. New localities (n = 12) with D. reticulatus occurrence have been found in southern Latvia.