The Prevalence of Tick-Borne Encephalitis Virus in Wild Rodents Captured in Tick-Borne Encephalitis Foci in Highly Endemic Lithuania.

Wild rodents are considered to be one of the most important TBEV-amplifying reservoir hosts; therefore, they may be suitable for foci detection studies. To investigate the effectiveness of viral RNA detection in wild rodents for suspected TBEV foci confirmation, we trapped small rodents (n = 139) in various locations in Lithuania where TBEV was previously detected in questing ticks. Murine neuroblastoma Neuro-2a cells were inoculated with each rodent sample to maximize the chances of detecting viral RNA in rodent samples. TBEV RNA was detected in 74.8% (CI 95% 66.7-81.1) of the brain and/or internal organ mix suspensions, and the prevalence rate increased significantly following sample cultivation in Neuro-2a cells. Moreover, a strong correlation (r = 0.88; p < 0.05) was found between the average monthly air temperature of rodent trapping and the TBEV RNA prevalence rate in cell culture isolates of rodent suspensions, which were PCR-negative before cultivation in cell culture. This study shows that wild rodents are suitable sentinel animals to confirm TBEV foci. In addition, the study results demonstrate that sample cultivation in cell culture is a highly efficient method for increasing TBEV viral load to detectable quantities.

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.

Prevalence and Diversity of Haemotropic Mycoplasma Species in Cats and Their Ectoparasites (Fleas and Ticks).

Mycoplasma spp. pathogens frequently cause chronic and acute diseases in cats. The aim of the present study was to investigate the presence and genetic diversity of Mycoplasma spp. in cats and their ectoparasites using PCR and sequence analysis of the 16S rRNA gene. Blood samples were collected from 541 domestic and stray cats in Lithuania. Ectoparasites (153 fleas and 321 ticks) were collected from owned domestic cats that live both outdoors and indoors. Mycoplasma spp. were detected in 7.2% of cat blood samples and 4.4% of Ctenocephalides felis fleas. The sequence analysis revealed the presence of Mycoplasma haemofelis in 1.1% of cats and 'Candidatus Mycoplasma haematominutum' in 4.8% of cats. Ct. felis fleas harboured M. haemofelis. To the best of the authors' knowledge, this is the first report on the prevalence and molecular characterisation of Mycoplasma bacteria in cats in Lithuania and cat fleas in the Baltic States.

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.

Anaplasma phagocytophilum in European bison (Bison bonasus) and their ticks from Lithuania and Poland.

The increasing population of European bison (Bison bonasus) can contribute to the prevalence of zoonotic pathogens. The aim of the present study was to assess the presence of A. phagocytophilum infection in European bison tissues as well as ticks removed from European bison in Lithuania and Poland. A further objective of this work was to compare the detected A. phagocytophilum strains. A total of 85 tissue samples (spleen) of European bison and 560 ticks belonging to two species, Ixodes ricinus (n = 408) and Dermacentor reticulatus (n = 152) were tested. DNA of A. phagocytophilum was detected based on RT-PCR in 40% of the European bison samples, 8.8% of the I. ricinus and 5.9% of the D. reticulatus ticks. Analysis of the obtained partial 16S rRNA gene sequences of A. phagocytophilum revealed the presence of three variants with two polymorphic sites. Furthermore, phylogenetic analysis with partial msp4 gene sequences grouped A. phagocytophilum variants into three clusters. This study revealed that the groEL gene sequences of A. phagocytophilum from European bison and their ticks grouped into ecotype I and only one sequence from Lithuanian European bison belonged to ecotype II. The results of the present study indicated that European bison may play a role as a natural reservoir of A. phagocytophilum.

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.

Association between Milk Electrical Conductivity Biomarkers with Lameness in Dairy Cows.

Early identification of lameness at all phases of lactation improves milk yield and reduces the incidence of mastitis in the herd. According to the literature we hypothesized that there are associations of electrical conductivity variables of milk flow with lameness in dairy cows. The aim of this study was to determine if blood cortisol and electrical conductivity in the milk flow phases correlate with each other and whether they are related to cow lameness. On one farm, out of 1500 cows, 64 cows with signs of lameness and 56 healthy cows were selected with an average of 2.8 lactations and 60 days in the postpartum period. A local veterinarian who specializes in hoof care treatments identified and scored lameness. During evening milking, the milk flow of all 120 cows was measured using electronic milk flow meters (Lactocorder®, WMB AG, Balgache, Switzerland). Before each milking, two electronic mobile milk flow meters (Lactocorders) were mounted between the milking apparatus and the milking tube to take measurements. We found that the average cortisol concentration in the blood of the studied cows was significantly correlated with the laminitis score. Results of this study indicate that the number of non-lame cows with a milk electrical conductivity level of <6 mS/cm even reached 90.8−92.3% of animals. Milk electrical conductivity indicators ≥ 6 mS/cm were determined in 17.8−29.0% more animals in the group of lame cows compared to the group of non-lame cows. According to our study, we detected that blood cortisol concentration had the strongest positive correlation with milk electrical conductivity indicators. Cows with a greater lameness score had a higher cortisol content and milk conductivity.

Bartonella spp. detection in laelapid (Mesostigmata: Laelapidae) mites collected from small rodents in Lithuania.

The genus Bartonella contains facultative Gram-negative intracellular bacteria from the family Bartonellaceae that can cause diseases in humans and animals. Various Bartonella species have been detected in rodents' ectoparasites, such as fleas, ticks, mites, and lice. However, the role of laelapid mites (Mesostigmata: Laelapidae) as carriers of Bartonella spp. needs to be confirmed. We aimed to investigate the presence of Bartonella spp. in laelapid mites collected from small rodents in Lithuania using real-time PCR targeting the transfer-messenger RNA/tmRNA (ssrA) gene and to characterize Bartonella strains using nested PCR and sequence analysis of the 16S-23S rRNA intergenic transcribed spacer region (ITS). A total of 271 laelapid mites of five species (Laelaps agilis, Haemogamasus nidi, Eulaelaps stabularis, Myonyssus gigas, and Hyperlaelaps microti) were collected from five rodent species (Apodemus flavicollis, Apodemus agrarius, Clethrionomys glareolus, Micromys minutus, and Microtus oeconomus) during 2015-2016. Bartonella DNA was detected in three mite species L. agilis, M. gigas, and Hg. nidi with an overall prevalence of 11.4%. Sequence analysis of the 16S-23S rRNA ITS region revealed the presence of Bartonella taylorii in L. agilis, Hg. nidi, and M. gigas, and Bartonella grahamii in L. agilis. Our results suggest that laelapid mites are involved in the maintenance of rodent-associated Bartonella spp. in nature. To the best of the authors' knowledge, this is the first study to demonstrate the presence of Bartonella spp. DNA in laelapid mites from small rodents.

Changes in the Genetic Structure of Lithuania's Wild Boar (Sus scrofa) Population Following the Outbreak of African Swine Fever.

The emergence of African swine fever (ASF) in Lithuania and its subsequent persistence has led to a decline in the population of wild boar (Sus scrofa). ASF has been spreading in Lithuania since its introduction, therefore it is important to understand any genetic impact of ASF outbreaks on wild boar populations. The aim of this study was to assess how the propensity for an outbreak has shaped genetic variation in the wild boar population. A total of 491 wild boar samples were collected and genotyped using 16 STR markers. Allele richness varied between 15 and 51, and all SSR loci revealed a significant deviation from the Hardy-Weinberg equilibrium. Fixation indices indicated a significant reduction in heterozygosity within and between subpopulations. PCoA and STRUCTURE analysis demonstrated genetic differences between the western region which had had no outbreaks (restricted zone I) and the region with ASF infection (restricted zones II and III). It is concluded that environmental factors may play a particular role in shaping the regional gene flow and influence the genetic structure of the wild boar population in the region with ASF outbreaks.

Molecular diversity of Paenibacillus larvae strains isolated from Lithuanian apiaries.

Paenibacillus larvae bacterium is known to be the causative agent of American foulbrood (AFB), a widespread, highly contagious and fatal disease in honey bees (Apis mellifera). There are four genotypes of Paenibacillus larvae that are named after their enterobacterial repetitive consensus (ERIC), and a fifth ERIC genotype has recently been found. In this study, a total of 108 independent P. larvae isolates from different geographical regions in Lithuania collected between 2011 and 2021 were investigated by molecular methods. The aims of this study were to detect which enterobacterial repetitive intergenic consensus (ERIC) genotype is the most common in Lithuania apiaries, identify and differentiate subtypes of the defined genotype by using multiple-locus variable number of tandem-repeat analysis (MLVA), and review how bacterial molecular diversity has changed over time in different parts of Lithuania. The obtained molecular analysis results showed that 100% of P. larvae bacterial isolates from Lithuania belong to the ERIC I genotype and can be differentiated to nine different subtypes by using the MLVA and capillary electrophoresis methods.

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.

Subclinical Mastitis Detected during the Last Gestation Period Can Increase the Risk of Stillbirth in Dairy Calves.

We hypothesized that subclinical mastitis detected during the last gestation period can increase the risk of stillbirth in dairy calves. The aim was to investigate the relation of subclinical mastitis detected during the last gestation period and its pathogens with the stillbirth of calves. Cows from the 210th day of pregnancy were selected for the study. They were divided into two groups: the first group-subclinical mastitis was confirmed on the farm by the California mastitis test (CMT); the second group of cows-mastitis was not confirmed by the CMT test. Groups of cows were compared according to the results of their calving-the number of stillborn calves. A stillborn calf was defined as a calf that dies at birth or within the first 24 h after calving, following a gestation period of 260 days. Our results suggest that decreasing the incidence of subclinical mastitis during the last gestation period (from the 210th day of pregnancy) can decrease the risk of stillbirth in dairy calves. Further, it is important to identify the pathogen because the highest risk of stillbirth was found in cows with mastitis caused by Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, pathogenic Staphylococci and other Streptococci. Cows at the first calving had a 1.38-1.65-times higher risk of having stillborn calves than cows of parity ≥ 2. From a practical point, veterinarians and farmers can consider the effect of subclinical mastitis during late gestation on the risk of stillbirth and it could help for strategies of optimizing reproductive performance in dairy cows.

Babesiosis in Southeastern, Central and Northeastern Europe: An Emerging and Re-Emerging Tick-Borne Disease of Humans and Animals.

There is now considerable evidence that in Europe, babesiosis is an emerging infectious disease, with some of the causative species spreading as a consequence of the increasing range of their tick vector hosts. In this review, we summarize both the historic records and recent findings on the occurrence and incidence of babesiosis in 20 European countries located in southeastern Europe (Bosnia and Herzegovina, Croatia, and Serbia), central Europe (Austria, the Czech Republic, Germany, Hungary, Luxembourg, Poland, Slovakia, Slovenia, and Switzerland), and northern and northeastern Europe (Lithuania, Latvia, Estonia, Iceland, Denmark, Finland, Sweden, and Norway), identified in humans and selected species of domesticated animals (cats, dogs, horses, and cattle). Recorded cases of human babesiosis are still rare, but their number is expected to rise in the coming years. This is because of the widespread and longer seasonal activity of Ixodes ricinus as a result of climate change and because of the more extensive use of better molecular diagnostic methods. Bovine babesiosis has a re-emerging potential because of the likely loss of herd immunity, while canine babesiosis is rapidly expanding in central and northeastern Europe, its occurrence correlating with the rapid, successful expansion of the ornate dog tick (Dermacentor reticulatus) populations in Europe. Taken together, our analysis of the available reports shows clear evidence of an increasing annual incidence of babesiosis across Europe in both humans and animals that is changing in line with similar increases in the incidence of other tick-borne diseases. This situation is of major concern, and we recommend more extensive and frequent, standardized monitoring using a "One Health" approach.

Pan-European phylogeography of the European roe deer (Capreolus capreolus).

To provide the most comprehensive picture of species phylogeny and phylogeography of European roe deer (Capreolus capreolus), we analyzed mtDNA control region (610 bp) of 1469 samples of roe deer from Central and Eastern Europe and included into the analyses additional 1541 mtDNA sequences from GenBank from other regions of the continent. We detected two mtDNA lineages of the species: European and Siberian (an introgression of C. pygargus mtDNA into C. capreolus). The Siberian lineage was most frequent in the eastern part of the continent and declined toward Central Europe. The European lineage contained three clades (Central, Eastern, and Western) composed of several haplogroups, many of which were separated in space. The Western clade appeared to have a discontinuous range from Portugal to Russia. Most of the haplogroups in the Central and the Eastern clades were under expansion during the Weichselian glacial period before the Last Glacial Maximum (LGM), while the expansion time of the Western clade overlapped with the Eemian interglacial. The high genetic diversity of extant roe deer is the result of their survival during the LGM probably in a large, contiguous range spanning from the Iberian Peninsula to the Caucasus Mts and in two northern refugia.

First Molecular Detection of Bartonella bovis and Bartonella schoenbuchensis in European Bison (Bison bonasus).

Bartonella bacteria infect the erythrocytes and endothelial cells of mammalians. The spread of the Bartonella infection occurs mainly via bloodsucking arthropod vectors. Studies on Bartonella infection in European bison, the largest wild ruminant in Europe, are lacking. They are needed to clarify their role in the maintenance and transmission of Bartonella spp. The aim of this study was to investigate the presence of the Bartonella pathogen in European bison and their ticks in Lithuania. A total of 38 spleen samples from bison and 258 ticks belonging to the Ixodes ricinus and Dermacentor reticulatus species were examined. The bison and tick samples were subjected to ssrA, 16S-23S rRNA ITS, gltA, and rpoB partial gene fragment amplification using various variants of PCR. Bartonella DNA was detected in 7.9% of the tissue samples of European bison. All tick samples were negative for Bartonella spp. The phylogenetic analysis of 16S-23S rRNA ITS, gltA, and rpoB partial gene fragment revealed that European bison were infected by B. bovis (2.6%) and B. schoenbuchensis (5.3%). This is the first report addressing the occurrence of B. bovis and B. schoenbuchensis in European bison in Europe.

Impact of tick-borne Anaplasma phagocytophilum infections in calves of moose (Alces alces) in southern Norway.

The Gram-negative, obligate intracellular tick-transmitted pathogen Anaplasma phagocytophilum can cause acute febrile diseases in humans and domestic animals. The expansion of the tick Ixodes ricinus (Linnaeus, 1758) in northern Europe due to climate change is of serious concern for animal and human health. The aim of the present study was to investigate the impact of A. phagocytophilum infection in moose Alces alces (Linnaeus) calves by evaluating the carcass weights of infected and non-infected animals and examining animal tissues samples for co-infections with either species of Babesia Starcovici, 1893 or bacteria of the genus Bartonella. The carcasses of 68 free-ranging moose calves were weighed by hunters during the hunting seasons from 2014 to 2017 in two regions in southern Norway and spleen samples were collected. Anaplasma phagocytophilum was detected in moose sampled from locations infected with ticks with a prevalence of 82% (n = 46). The carcass weights of A. phagocytophilum-infected calves (n = 46) and non-infected (n = 22) calves were compared. Although the average weight of infected calves (45.6 kg) was lower than that of non-infected calves (46.5 kg), the difference was not statistically significant. Three different variants of the bacterium 16S rRNA gene were identified. The average weight of animals infected with variant I was 49.9 kg, whereas that of animals infected with variant III was 42.0 kg, but the difference was not statistically significant (p = 0.077). Co-infections of A. phagocytophilum with Bartonella spp. or with Babesia spp. were found in 20 and two calves, respectively. A triple infection was found in two calves. Sequence analysis of the 18S rRNA gene of Babesia-positive samples revealed the presence of Babesia cf. odocoilei (Emerson et Wright, 1970). Strains of Bartonella closely related to Bartonella bovis (Bermond, Boulouis, Heller, Laere, Monteil, Chomel, Sander, Dehio et Piemont, 2002) were identified based on phylogenetic analysis of the gltA and rpoB genes. The loss of body mass in moose calves in the tick-infected site was probably influenced by multiple factors.

The effects of habitat fragmentation on the genetic structure of wild boar (Sus scrofa) population in Lithuania.

BACKGROUND: Wild boar (Sus scrofa) is a widely distributed ungulate whose success can be attributed to a variety of ecological features. The genetic variation and population structure of Lithuania's wild boar population have not yet been thoroughly studied. The purposes of this study were to investigate the genetic diversity of S. scrofa and assess the effects of habitat fragmentation on the population structure of wild boar in Lithuania. A total of 96 S. scrofa individuals collected from different regions of Lithuania were genotyped using fifteen microsatellite loci. RESULTS: The microsatellite analysis of the wild boars indicated high levels of genetic diversity within the population. Microsatellite markers showed evidence of a single panmictic wild boar population in Lithuania according to STRUCTURE's highest average likelihood, which was K = 1. This was supported by pairwise Fst values and AMOVA, which indicated no differentiation between the four sampling areas. The results of the Mantel test revealed a weak isolation by distance and geographic diversity gradients that persisted between locations. Motorway fencing and heavy traffic were not an effective barrier to wild boar movement. CONCLUSIONS: There was limited evidence of population genetic structure among the wild boar, supporting the presence of a single population across the study area and indicating that there may be no barriers hindering wild boar dispersal across the landscape. The widespread wild boar population in Lithuania, the high level of genetic variation observed within subpopulations, and the low level of variation identified between subpopulations suggest migration and gene flow between locations. The results of this study should provide valuable information in future for understanding and comparing the detailed structure of wild boar population in Lithuania following the outbreak of African swine fever.

Bartonella Infections in Cats and Cat Fleas in Lithuania.

Bartonella are vector-borne parasitic bacteria that cause zoonotic infections in humans. One of the most common infections is cat-scratch disease caused by Bartonella henselae and Bartonella clarridgeiae. Cats are the major reservoir for these two species of bacteria, while cat fleas are vectors for the transmission of infection agents among cats. The aim of the present study was to investigate the presence of Bartonella infections in stray and pet cats and in cat fleas in Lithuania. Blood samples were taken from 163 cats presented in pet clinics and animal shelters. A total of 102 fleas representing two species, Ctenocephalides felis and Ctenocephalides canis, were collected from 12 owned cats that live both outdoors and indoors. Bartonella DNA in samples was detected using a nested PCR targeting the 16S-23S rRNA intergenic spacer (ITS) region. Bartonella DNA was detected in 4.9% (8/163) of the cats and 29.4% (30/102) of the fleas. Sequence analysis of the ITS region showed that the cats and fleas were infected with B. henselae, B. clarridgeiae and Bartonella sp., closely related to B. schoenbuchensis. This study is the first report on the prevalence and molecular characterization of Bartonella spp. in cats and cat fleas in Lithuania.

Bulk Milk Tank Samples Are Suitable to Assess Circulation of Tick-Borne Encephalitis Virus in High Endemic Areas.

A reliable surveillance strategy of tick-borne encephalitis virus (TBEV) is necessary to ensure adequate disease control measures. However, current approaches assessing geographical TBEV circulation are ineffective or have significant limitations. In this study we investigated a total of 1363 goat and 312 sheep bulk tank milk samples for the presence of TBEV. Samples were collected from systematically selected farms in Lithuania every 4-5 days from April to November in 2018 and 2019. To validate results, we additionally tested 2685 questing ticks collected in the vicinity of milk collection sites. We found 4.25% (95% CI 3.25-5.47) and 4.48% (95% CI 2.47-7.41) goat and sheep milk samples to be positive for TBEV, respectively. Furthermore, geographical distribution of TBEV in milk samples coincided with the known TBE endemic zone and was correlated with incidence of TBE in humans in 2019. When sampling time coincides, TBEV detection in milk samples is as good a method as via flagged ticks, however bulk milk samples can be easier to obtain more frequently and regularly than tick samples. The minimal infectious rate (MIR) in ticks was 0.34% (CI 95% 0.15-0.64). Therefore, our results confirm that testing milk serves as a valuable tool to investigate the spatial distribution of TBEV at higher resolution and lower cost.

Molecular detection of Babesia spp. in European bison (Bison bonasus) and their ticks.

Babesia spp. are tick-borne haemoparasites that infect a wide range of domestic and wild mammals. Free-ranging ungulates are considered to be important reservoir hosts of Babesia parasites. The European bison (Bison bonasus) is a large and rare ungulate species, reintroduced into the forests of Central Europe after an absence of several decades. Owing to their protected status, studies of tick-borne pathogens in European bison have so far been rare and fragmented. The aim of this study was to investigate the presence of Babesia infection in free-ranging and captive herds of European bison and their ticks. Tissue samples obtained from 37 European bison individuals and 242 ticks belonging to two species, Ixodes ricinus and Dermacentor reticulatus, collected from bison were subjected to PCR analysis of the 18S rRNA gene followed by sequencing. Babesia spp. were detected in 8% of the samples from European bison and in 11% of the ticks. Sequence analysis of partial 18S rRNA gene indicated the presence of B. divergens and B. capreoli in European bison, while B. divergens, B. microti and B. venatorum were detected in ixodid ticks. To the best of authors' knowledge, this is the first molecular detection and characterization of Babesia spp. in European bison and their ticks.