Candidatus Neoehrlichia mikurensis and Hepatozoon sp. in voles (Microtus spp.): occurrence and evidence for vertical transmission.

Candidatus Neoehrlichia mikurensis (CNM) and Hepatozoon spp. are important vector-borne parasites of humans and animals. CNM is a relatively recently discovered pathogen of humans. Hepatozoon are parasites of reptiles, amphibians and mammals, commonly found in rodents and carnivores worldwide. The present study aimed to determine the prevalence of CNM and Hepatozoon spp. in three species of Microtus and to assess the occurrence of vertical transmission in naturally-infected voles. Molecular techniques were used to detect pathogen DNA in blood and tissue samples of captured voles and their offspring. The prevalence of CNM in the vole community ranged 24-47% depending on Microtus species. The DNA of CNM was detected in 21% of pups from three litters of six infected Microtus dams (two Microtus arvalis and one M. oeconomus) and in 3/45 embryos (6.6%) from two litters of eight CNM-infected pregnant females. We detected Hepatozoon infection in 14% of M. arvalis and 9% of M. oeconomus voles. Hepatozoon sp. DNA was detected in 48.7% of pups from seven litters (6 M. arvalis and 1 M. oeconomus) and in two embryos (14.3%) obtained from one M. arvalis litter. The high prevalence of CNM infections in the Microtus spp. community may be a result of a relatively high rate of vertical transmission among naturally infected voles. Vertical transmission was also demonstrated for Hepatozoon sp. in M. arvalis and M. oeconomus. Our study underlines the significance of alternative routes of transmission of important vector-borne pathogens.

Contribution of tick-borne diseases to mortality in juvenile free-living cervids.

INTRODUCTION AND OBJECTIVE: Reports on tick-borne infections in free-living juvenile animals and their impact on survival of cervids in nature are lacking. The aim of the study was to detect and identify the Babesia and Anaplasma phagocytophilum species/ecotypes that may have contributed to the death of juvenile animals from a wildlife rescue centre in spring 2020. MATERIAL AND METHODS: PCR amplification and sequencing of two genetic markers (18S rDNA and cox1 for Babesia, 16S rDNA and groEL for A. phagocytophilum) were used for screening eleven samples derived from juvenile animals which died in a rescue centre (seven roe deer Capreolus capreolus, one elk Alces alces, one red squirrel Sciurus vulgaris, one European beaver Castor fiber, one red fox Vulpes vulpes). Phylogenetic analysis of full-length 18S rDNA sequence was performed to enable differentiation between two closely-related species infecting wild ungulates, Babesia capreoli and Babesia divergens (zoonotic). RESULTS: The occurrence of the typical SNPs of B. capreoli at two discriminating positions in the 18S rRNA gene allowed identification of B. capreoli infection in a roe deer calf. In two calves, Anaplasma phagocytophilum ecotype 2 was identified, including the same calf co-infection. No Babesia DNA was amplified in an elk calf treated for babesiosis. Splenomegaly was recorded in roe deer calves with A. phagocytophilum and A. phagocytophilum + B. capreoli infection. CONCLUSIONS: The study revealed that tick-borne infections are common among juvenile, free-living cervids and may contribute to their mortality.

Tick-Borne Pathogens, Babesia spp. and Borrelia burgdorferi s.l., in Sled and Companion Dogs from Central and North-Eastern Europe.

Ticks are important vectors of numerous pathogens of medical and veterinary significance. The aim of the current study was to determine the prevalence of Babesia spp. and Borrelia burgdorferi s.l. in sled and pet dogs from Central and North-Eastern Europe. Neither Babesia spp. nor Borrelia burgdorferi s.l. infections were detected in sled dogs from seven countries (Poland, Lithuania, Latvia, Estonia, Belarus, Russia and Finland). The DNA of Babesia spp. was detected in 100% of symptomatic and 5.4% of asymptomatic pet dogs from Poland. Similarly, the DNA of Babesia spp. was identified in 82% of symptomatic and 3.8% of asymptomatic pet dogs from Ukraine. The DNA of Borrelia burgdorferi s.l. was detected in 4.4% of pet dogs. Molecular typing confirmed the presence of Babesia canis and Borrelia burgdorferi sensu stricto (s.s.) in selected samples. Four dogs were co-infected by B. canis and Borrelia burgdorferi s.l. Tick-borne pathogens constitute a serious health threat to pet dogs in Central and South-Eastern Europe, but were not observed among sled dogs from the same region of Europe nor in the Baltic countries.

Borrelia miyamotoi infection in Apodemus spp. mice populating an urban habitat (Warsaw, Poland).

The two of three species of city-inhabiting (Warsaw, Poland) Apodemus spp. mice were showed to be infected with Borrelia miyamotoi, spirochete bacterium considered to be a tick-borne relapsing fever (TBRF) disease causative agent. The pathogen infection was ascertained based on bacterium DNA presence in a blood (obtained from the lateral tail vein) of the mice, using the nested polymerase chain reaction (PCR) technique. The B. miyamotoi carriers populated not only green spaces situated in the suburban areas but also recreational parks and lawns in the city centre proper. Moreover, we found no significant differences in the infection rate depending on mouse species. The total prevalence of infection at the level of 5.6% suggests the potential role of urban-settled rodents in the spreading of the tick-borne zoonosis, which pose high risk to public health.

Ectoparasites of red foxes (Vulpes vulpes) with a particular focus on ticks in subcutaneous tissues.

In the present study, we identified the ectoparasite communities of red foxes in three regions of Poland that encompassed two endemic regions for the occurrence of Dermacentor reticulatus, as well as a region that is free of this tick species ('gap' area). Our study sites were selected to enable the role of foxes as hosts for juvenile (nest dwelling) and adult (exophilic) D. reticulatus ticks to be determined, and to assess their contribution to the spread of this important vector of Babesia canis. We compared also ectoparasite communities between adult foxes with those of fox cubs. Finally, we carried out a systematic search for subcutaneous ticks determining their prevalence and abundance. In 2016-2018, 366 adult foxes and 25 live-trapped cubs were examined for ectoparasites. Ectoparasites were identified based on morphological features, PCR amplification and sequencing. The total prevalence of ectoparasites was higher in cubs (68%) than in adults (62.8%). In adults, 15 parasite species were recorded, including four tick species, seven flea species, scabies, and one Anopluran species each in the genera Felicola and Lipoptena. In cubs, six ectoparasite species were found, including Ixodes kaiseri, a species not found in adults. Although Ixodes ricinus and D. reticulatus were the dominant tick species on adult foxes, no D. reticulatus ticks were found on cubs. Subcutaneous ticks were common (38%) and abundant in all areas. Molecular analysis of subcutaneous nodules allowed the identification of 17 I. ricinus and five D. reticulatus. In conclusion, red foxes play a minor role as hosts of D. reticulatus.

Different Competence as a Lyme Borreliosis Causative Agent Reservoir Found in Two Thrush Species: The Blackbird (Turdus merula) and the Song Thrush (Turdus philomelos).

Passerine birds are significantly involved in the dissemination of Borreliella spp. bacteria (formerly Borrelia burgdorferi sensu lato complex), the causative agent of most common and important tick-spread disease Lyme borreliosis. Among several dozen investigated passerine bird species, thrushes (Turdidae) have been reported as a relatively good pathogen reservoirs and disseminators. The principal aim of the study was to identify the differences in Borreliella spp. reservoir competence between two widespread and showing similar behavior thrush species. A total of 157 Ixodes ricinus ticks (19 larvae, 138 nymphs) were collected from 26 blackbirds (Turdus merula) and 20 song thrushes (Turdus philomelos) living in the same forest habitat (northeastern Poland). All, at least partially engorged ectoparasites, were tested for the presence of Borreliella spp. DNA using the nested-PCR technique. No significant difference of tick prevalence was found, with 88.5% blackbirds and 70% of song thrushes infested. Screening for Borreliella spp. in ticks revealed that both tick infection prevalence (49.2% vs. 18.9%) and mean number of infected individuals engorging on birds (2.27 vs. 0.35) were higher in blackbirds. Both the investigated thrush species presumably could participate in the pathogen circulation, although with different efficiencies. The greater reservoir competence of blackbirds suggests the differentiated dynamics of host-pathogen interactions among selected species, and consequently their potentially disparate role in the ecology of Lyme borreliosis.

Rickettsiales Occurrence and Co-occurrence in Ixodes ricinus Ticks in Natural and Urban Areas.

Bacteria of Rickettsiaceae and Anaplasmataceae families include disease agents spread by Ixodes ricinus ticks, the most common tick vector in Europe. The aim of the study was to compare the prevalence and co-infection prevalence of particular tick-transmitted Rickettsiales members: Rickettsia spp. (further referred as Rs), Anaplasma phagocytophilum (Ap), and "Candidatus Neoehrlichia mikurensis" (CNM) in I. ricinus ticks in two types of areas, different in terms of human impact: natural and urban. Using additional data, we aimed at investigating co-occurrence of these Rickettsiales with Borreliella spp. A total of 4189 tick specimens, 2363 from the urban area (Warsaw park and forests) and 1826 from the natural area (forests and park in the vicinity of National Parks), were tested for the presence of Rickettsiales DNA by PCRs. The prevalence of selected Rickettsiales was twice higher in urban than natural areas (13.2% vs. 6.9%, respectively). In total ticks, the prevalence of Rs, Ap, and CNM was 6.5%, 5.3%, and 3.6% in urban areas vs. 4.4%, 1.1%, and 2.1% in natural areas, respectively. Co-infections of Rickettsiales were also more prevalent in urban areas (2.6% vs. 0.3%, respectively). The most common Rs was R. helvetica; also R. monacensis and novel "Candidatus Rickettsia mendelii" were detected. Positive association between Ap and CNM infections was discovered. Rickettsiales bacteria occurrence was not associated with Borreliella occurrence, but co-infections with these two groups were more common in ticks in urban areas. In conclusion, three groups of Rickettsiales constituted the important part of the tick pathogen community in Poland, especially in the urbanized central Poland (Mazovia). In the Warsaw agglomeration, there is a greater risk of encountering the I. ricinus tick infected with Rickettsiales and co-infected with Lyme spirochaetes, in comparison to natural areas. This finding raises the question whether cities might in fact be the hot spots for TBDs.

Bartonella infections in three species of Microtus: prevalence and genetic diversity, vertical transmission and the effect of concurrent Babesia microti infection on its success.

BACKGROUND: Bartonella spp. cause persistent bacterial infections in mammals. Although these bacteria are transmitted by blood-feeding arthropods, there is also evidence for vertical transmission in their mammalian hosts. We aimed to determine: (i) the prevalence and diversity of Bartonella spp. in a Microtus spp. community; (ii) whether vertical transmission occurs from infected female voles to their offspring; (iii) the effect of concurrent Babesia microti infection on the success of vertical transmission of Bartonella; and (iv) the impact of congenital infection on pup survival. RESULTS: We sampled 124 Microtus arvalis, 76 Microtus oeconomus and 17 Microtus agrestis. In total, 115 embryos were isolated from 21 pregnant females. In the following year 11 pregnant females were kept until they had given birth and weaned their pups (n = 62). Blood smears and PCR targeting the Bartonella-specific rpoB gene fragment (333bp) were used for the detection of Bartonella. Bartonella DNA was detected in 66.8% (145/217) of the wild-caught voles. Bartonella infection was detected in 81.8% (36/44) of pregnant female voles. Bartonella-positive individuals were identified among the embryos (47.1%; 40/85) and in 54.8% (34/62) of pups. Congenitally acquired Bartonella infections and co-infection with B. microti had no impact on the survival of pups over a 3-week period post partum. Among 113 Bartonella sequences, four species were detected: Bartonella taylorii, Bartonella grahamii, Bartonella doshiae and a Bartonella rochalimae-like genotype. Bartonella taylorii clade B was the dominant species in wild-caught voles (49%), pregnant females (47%), their embryos (85%), dams (75%) and pups (95%). CONCLUSIONS: High prevalence of Bartonella spp. infection maintained in Microtus spp. community is followed by a high rate of vertical transmission of several rodent species of Bartonella in three species of naturally infected voles, M. arvalis, M. oeconomus and M. agrestis. Congenitally acquired Bartonella infection does not affect the survival of pups. Co-infection with B. microti does not affect the effectiveness of the vertical transmission of Bartonella in voles. Bartonella taylorii clade B was found to be the dominant species in wild-caught voles, including pregnant females and dams, and in their offspring, and was also found to be the most successful in vertical transmission.

Ticks and the city - are there any differences between city parks and natural forests in terms of tick abundance and prevalence of spirochaetes?

BACKGROUND: Ixodes ricinus ticks are commonly encountered in either natural or urban areas, contributing to Lyme disease agents Borreliella [(Borrelia burgdorferi (sensu lato)] spp. and Borrelia miyamotoi enzootic cycles in cities. It is an actual problem whether urbanization affects pathogen circulation and therefore risk of infection. The aim of the study was to evaluate main tick-borne disease risk factors in natural, endemic areas of north-east (NE) Poland (Bialowieza) and urban areas of central Poland (Warsaw), measuring tick abundance/density, prevalence of infection with spirochaetes and diversity of these pathogens in spring-early summer and late summer-autumn periods between 2012 and 2015. METHODS: Questing I. ricinus ticks were collected from three urban sites in Warsaw, central Poland and three natural sites in Bialowieza, NE Poland. A total of 2993 ticks were analyzed for the presence of Borreliella spp. and/or Borrelia miyamotoi DNA by PCR. Tick abundance was analyzed by General Linear Models (GLM). Prevalence and distribution of spirochaetes was analyzed by Maximum Likelihood techniques based on log-linear analysis of contingency tables (HILOGLINEAR). Species typing and molecular phylogenetic analysis based on the sequenced flaB marker were carried out. RESULTS: Overall 4617 I. ricinus ticks were collected (2258 nymphs and 2359 adults). We report well established population of ticks in urban areas (10.1 ± 0.9 ticks/100 m2), as in endemic natural areas with higher mean tick abundance (16.5 ± 1.5 ticks/100 m2). Tick densities were the highest in spring-early summer in both types of areas. We observed no effect of the type of area on Borreliella spp. and B. miyamotoi presence in ticks, resulting in similar prevalence of spirochaetes in urban and natural areas [10.9% (95% CI: 9.7-12.2%) vs 12.4% (95% CI: 10.1-15.1%), respectively]. Prevalence of spirochaetes was significantly higher in the summer-autumn period than in the spring-early summer [15.0% (95% CI: 12.8-17.5%) vs 10.4% (95% CI: 9.2-11.6%), respectively]. We have detected six species of bacteria present in both types of areas, with different frequencies: dominance of B. afzelii (69.3%) in urban and B. garinii (48.1%) in natural areas. Although we observed higher tick densities in forests than in maintained parks, the prevalence of spirochaetes was significantly higher in the latter [9.8% (95% CI: 8.6-11.0%) vs 17.5% (95% CI: 14.4-20.5%)]. CONCLUSIONS: Surprisingly, a similar risk of infection with Borreliella spp. and/or B. miyamotoi was discovered in highly- and low-transformed areas. We suggest that the awareness of presence of these disease agents in cities should be raised.

Origins of recently emerged foci of the tick Dermacentor reticulatus in central Europe inferred from molecular markers.

The ornate dog tick Dermacentor reticulatus is vector of several blood parasites, including Babesia canis, a causative agent of babesiosis. The geographical range of D. reticulatus in Europe is discontinuous with a gap separating eastern and western macroregions. New foci observed in several locations in western and central Europe were considered an expansion of the western population, including foci in western Poland. In the present paper we used molecular markers to identify the origins of these foci, and we compared their genetic polymorphism to D. reticulatus collected in sites situated within the eastern population. The overall polymorphism in mt 16S rDNA was low, and all sites from the western population shared the same haplotype suggesting the expansion in this area. In the marker 5.8S rDNA-ITS2 we found no differences in polymorphism between sites from eastern Poland (eastern population), and newly emerged foci in western Poland considered a putative expansion zone of the western population. However, the sites from western Poland differed considerably from nearby German site. Our results show that foci in western Poland could not have originated from D. reticulatus from the western population, as previously thought. We found that the state border following river hinders considerably gene flow between adjacent sites what suggest that natural dispersal of D. reticulatus by wildlife is unlikely, and the emergence of new foci should rather be contributed to human-associated dispersal. We propose that livestock, and pets travelling with their owners are the most probable source of new foci, and they can easily transfer ticks within a country but not between countries.

Mapping of Dermacentor reticulatus expansion in Poland in 2012-2014.

Rapid expansion of the tick Dermacentor reticulatus (Fabricius) has been reported in many European countries. In Poland its range was limited to the area on the eastern side of the Vistula River up until the 1990s. However, new foci were recently discovered, while the centre of the country and mountain regions are believed to be free of this tick and are known as "the gap". A few unconfirmed factors, like unfavourable weather conditions, changes in land cover and absence of competent hosts have previously been reported as possible reasons for the absence of D. reticulatus in this area. Since D. reticulatus plays an important role for the maintenance and the circulation of tick-borne pathogens, we (1) determined its actual range in Poland, (2) monitored its expansion in 2012-2014 and (3) correlated abiotic conditions on its known range. Dragging was conducted in the area between the Vistula River and the western border of Poland in 2012-2014, along the three major Polish rivers and their tributaries. Temperature and humidity on the ground were recorded 4 times a day at a total of 32 sites. D. reticulatus was found in 21 new locations on the western side of the Vistula River and in 22 locations in western Poland. The presence of this tick species was confirmed for the first time in Wielkopolskie, Kujawsko-Pomorskie and Lódzkie voivodeships. Existence of 'the gap' was confirmed on a strip of land along the northwest-southeast axis (an area of approximately 151000km(2)), that extends between Zachodniopomorskie and Pomorskie in the North, and the mountain areas with foothills in the South, in Opolskie, Slaskie, Malopolskie and Podkarpackie voivodeships. This gap divides the tick population in Poland into two separated populations - Western and Eastern. The lowest abundance of ticks was found in the newly established foci in central and western Poland. Our study showed the need for the monitoring of the D. reticulatus expansion. Early detection of new foci is crucial for taking proper prevention measures.

Clinical and molecular features of one case of human infection with Anaplasma phagocytophilum from Podlaskie Province in eastern Poland.

The article focuses on the clinical and laboratory diagnosis of human granulocytic anaplasmosis (HGA) caused by Anaplasma phagocytophilum infection in one of 28 patients (3.6%; n=1/28 tested samples) with early Lyme borreliosis. The clinical and laboratory results of a 42-year-old patient fulfilled criteria of confirm anaplasmosis and suggest an acute stage of illness. The described case provides strong presumptive evidence that infection in this patient was acquired with a pathogenic strain of A. phagocytophilum through a tick bite. A positive DNA with PCR for A. phagocytophilum infection was sequenced and analyzed phylogenetically. Physicians should consider the possibility of anaplasmosis in patients with early Lyme borreliosis, and A. phagocytophilum should be considered as a differential diagnosis in all patients from an endemic region of potential high risk factors for tick-borne diseases.

Dominance of Dermacentor reticulatus over Ixodes ricinus (Ixodidae) on livestock, companion animals and wild ruminants in eastern and central Poland.

The most common tick species parasitizing animals in Poland are Ixodes ricinus and Dermacentor reticulatus. These tick species differ in their distribution, habitats, seasonal activity and host specificity. Ixodes ricinus is the most prevalent and widely distributed, whereas the range of D. reticulatus is limited to eastern and central parts of the country with several new foci in the middle-west and the west. However, as in many central European countries, the range of D. reticulatus is expanding, and some authors have correlated this expansion with an increasing number of available hosts. The aim of the present study was to determine the tick fauna on domestic and livestock animals in two areas endemic for I. ricinus and D. reticulatus and to compare the risk of infestation with different tick species in open and forest areas. Over a 14 month period, 732 ticks were collected from five host species including domestic animals (dogs and cats), livestock (cows and horses) and wildlife (European bison) in two areas, central and NE Poland, endemic for D. reticulatus. Three tick species were recorded: D. reticulatus (623 individuals; 85.1% of all collected ticks), I. ricinus (106 individuals; 14.5%) and three females of Ixodes hexagonus (0.4%) from a dog. Dermacentor reticulatus was the dominant tick species found on four host species and constituted 86, 81, 97 and 100% of all ticks from dogs, horses, cows and bison, respectively, and was collected from animals throughout the year, including during the winter. The common tick, I. ricinus, was the dominant tick collected from cats (94%). Fully-engorged, ready-for-reproduction females of D. reticulatus were collected from all host species. In May 2012, questing ticks were collected by dragging in forest or open habitats. The density of adult marsh ticks in open areas was around 2 ticks/100 m(2) in the majority of locations, with a maximum of 9.5 ticks/100 m(2). The density of adult I. ricinus was much lower in its typical habitat (forests: range 0.8-2.2 ticks/100 m(2)) between three and seven times lower than the density of D. reticulatus in its typical habitat. In regions endemic for marsh ticks, this tick species constitutes the main risk of tick infestation for livestock and dogs throughout the year. Livestock and companion animals are competent hosts for D. reticulatus, enabling the completion of the tick's life cycle. Anti-tick treatment should be adjusted to marsh tick seasonal activity and drug sensitivity.

Asymptomatic "Candidatus Neoehrlichia mikurensis" infections in immunocompetent humans.

In Europe, human infections with "Candidatus Neoehrlichia mikurensis" have mainly been restricted to immunocompromised patients. We report here the first cases of asymptomatic "Ca. Neoehrlichia mikurensis" infection in immunocompetent humans (5/316 [1.6%] were infected). Due to the potential threats of infections with "Ca. Neoehrlichia mikurensis" in healthy persons to the safety of the blood supply, further study of this phenomenon is required.

Rickettsiaceae and Anaplasmataceae infections in Ixodes ricinus ticks from urban and natural forested areas of Poland.

BACKGROUND: Ixodes ricinus is a major vector for a range of microbial pathogens and the most prevalent and widely distributed tick species on the European continent, occurring in both natural and urban habitats. Nevertheless, little is known about the relative density of ticks in these two ecologically distinct habitats and the diversity of tick-borne pathogens that they carry. METHODS: We compared densities of questing I. ricinus nymphs and adults in urban and natural habitats in Central and Northeastern Poland, assessed the prevalence and rate of co-infection with A. phagocytophilum, Rickettsia, Ehrlichia and 'Ca. Neoehrlichia spp.' in ticks, and compared the diversity of tick-borne pathogens using molecular assays (PCR). RESULTS: Of the 1325 adults and nymphs, 6.2% were infected with at least one pathogen, with 4.4%, 1.7% and less than 0.5% being positive for the DNA of Rickettsia spp., A. phagocytophilum, Ehrlichia spp. and Ca. N. mikurensis, respectively. Although tick abundance was higher in natural habitats, the prevalence of the majority of pathogens was higher in urban forested areas. CONCLUSION: We conclude that: (i) zoonotic genetic variants of A. phagocytophilum are widely distributed in the Polish tick population, (ii) although the diversity of tick borne pathogens was higher in natural habitats, zoonotic species/strains were detected only in urban forests, (iii) and we provide the first description of Ca. N. mikurensis infections in ticks in Poland.

The risk of vector-borne infections in sled dogs associated with existing and new endemic areas in Poland: Part 1: A population study on sled dogs during the racing season.

The achievements of sled dogs in competitions depend both on their training and on their health. Vector-borne infections may lead to anaemia, affect joints or heart muscle or even cause death. Between December 2009 and October 2010, one hundred and twenty six individual blood samples were collected from 26 sled dog kennels situated in different regions of Poland. The majority of samples were taken during the racing season (winter 2009/10). The prevalences of 3 vector-borne infections- including 2 'old pathogens' Anaplasma phagocytophilum and Babesia canis, and 'new pathogen' Hepatozoon canis-were estimated in sled dogs using PCR and nested PCR. Additionally, 25 serum samples originating from a subset of 3 kennels situated in a tick-borne encephalitis (TBE) endemic area (Mazowiecki region), were tested for antibodies against the tick-borne encephalitis virus (TBEV). Because of the recently reported occurrence of Dirofilaria repens in Central Poland and that of fatal cases of unknown aetiology in two of the kennels, blood samples collected from dogs at these kennels in 2010 and in February-May 2013 and from two unaffected kennels were checked for evidence of presence of this parasite. Babesia canis DNA was detected in 11 sled dogs (4 with clinical babesiosis, 7 asymptomatic; 8.7%) inhabiting mainly endemic regions of Poland (9/11 cases). Three serum samples originating from one location tested positive for TBEV antibodies (total seroprevalence: 3/25=12%, local seroprevalence: 3/12=25%). The risk of TBEV infection was associated with previous B. canis infections. Dirofilaria repens DNA was detected in 15 dogs (44%). Prevalence was especially high in two sled dog kennels situated near Grodzisk Mazowiecki (50-57%). No blood samples tested positive for A. phagocytophilum or H. canis DNA. The present study has established that the prevalence of vector-borne pathogens in working sled dogs is significant in the endemic regions and has justified the important role of surveillance of reservoir hosts in the epidemiology of TBE. Our results emphasize the need for regular monitoring for the presence of D. repens.