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Barbour-Stoenner-Kelly II (BSK-II) and BSK-H media were used for cultivation and isolation of fastidiousBorreliaspecies - the causative agents of Lyme borreliosis. Culture media have a limited shelf life and require adequate storage. Our goal was to assess how the growth ofBorreliawould be affected by prolonged storage of media and inadequate storage conditions (BSK-H stored at +4 degrees C for 2.5 years and BSK-II stored at -20 degrees C for 11 years). Growth of differentBorrelia afzelii,Borrelia garinii,Borrelia lusitaniaeandBorrelia valaisianastrains was assessed during 2 weeks of incubation at 33 degrees C. Monitored parameters included cell count per mL, morphology and motility. The results of this study have shown weaker growth of borrelia strains in BSK-H at +4 degrees C (median final cell number of 1.5 x 10(6)/mL) than in BSK-II at -20 degrees C (median final cell number of 7.75 x 10(6)/mL) and in fresh BSK-H media (median final cell number of 8.95 x 10(6)/mL). Duration of storage of media had no impact onBorreliamorphology and motility. Our results indicate that temperature of -20 degrees C is optimal for long-term storage of medium, BSK-II stored for 11 years provided effective support to growth ofBorreliaand may be employed for cultivation.

This study assessed the level of knowledge, attitudes, and practices (KAP) regarding tick-borne encephalitis virus (TBEV) and tick-borne diseases (TBDs) among different groups of people in Serbia. Professionally tick-exposed persons (PTEPs), health care workers (HCWs), and the general population (GP) were subjected to an anonymous, voluntary, online questionnaire using Microsoft Forms. A total of 663 questionnaire responses were collected (February–March 2021), while 642 were included in the analysis. The significant difference in knowledge in TBDs existed between GP and PTEPs, and HCWs (p < 0.001). The perception of risk-to-tick exposure and TBDs was generally high (42.4 (95% CI: 33.6–51.2) within GP, 44.9 (95% CI: 35.8–53.9) within PTEPs and 46.2 (95% CI: 38.0–54.5) within HCWs), while fear was low (13.7 (95% CI: 7.9–19.5) within GP, 12.6 (95% CI: 7.3–19.9) within PTEPs, and 13.5 (95% CI: 7.4–19.5) within HCWs). Protective practices differed across groups (F (2639) = 12.920, p < 0.001, η2 = 0.039), with both PTEPs (t = 3.621, Cohen d = 0.332, p < 0.001) and HCWs (t = 4.644, Cohen d = 0.468, p < 0.001) adhering to more protective practices than the GP, without differences between PTEPs and HCWs (t = 1.256, Cohen d = 0.137, p = 0.421). Further education about TBDs in Serbia is required and critical points were identified in this study.

Background The epidemiological status concerning many canine tick-borne diseases (TBDs) in Serbia is still insufficiently known. Objectives Our study aimed to investigate the presence of tick-borne pathogens of the family Anaplasmataceae and Hepatozoon spp., as a cause of illnesses accompanied by clinical signs that can occur in dogs with anaplasmosis, ehrlichiosis and hepatozoonosis. Methods Dogs are included in the study based on the presence of a minimum of three clinical and/or pathological findings that could be associated with anaplasmosis, ehrlichiosis and hepatozoonosis. During the study (April–October 2018), 11 dogs met the conditions to be included in the survey. Identification of the causative agent in the blood of diseased dogs was performed by conventional PCR followed by sequencing. Results The presence of the pathogens was confirmed in three animals (3/11, 27.3%). The presence of Ehrlichia canis was confirmed in 3-month-old female Rottweiler puppy, an 8-year old Miniature Schnauzer female was positive for Hepatozoon canis infection, while 4-year-old mixed breed male dog was co-infected with both mentioned pathogens. These are the first cases of autochthonous infection with E. canis and H. canis in dogs from Serbia confirmed by molecular methods. Conclusions The results of our study indicate the importance of molecular methods to establish a reliable diagnosis of TBDs. Also, the confirmed presence of causative agents of canine monocytic ehrlichiosis and hepatozoonosis in Serbia appeals to veterinary practitioners that it is necessary to exclude the presence of those diseases in suspicious dogs.

Wild canids are recognized as important reservoir hosts for several vector-borne pathogens and are a good sentinel species for epidemiological surveillance. Identifying the reservoir hosts for particular zoonotic pathogens in a specific area enables the implementation of adequate measures in order to prevent the occurrence of zoonotic diseases in humans and domestic animals. The present review aims to summarize the role of wild canids in the epidemiology of tick-borne zoonoses in Serbia based on previously published papers and to discuss the available data from an epidemiological point of view. The results of previously published studies indicate that wild canids are potential reservoir hosts for several zoonotic tick-borne pathogens in Serbia, and hosts for several tick species which are proven competent vectors. Based on molecular studies conducted so far, the presence of Anaplasma phagocytophilum, was confirmed in jackals while the presence of Candidatus Neoehrlichia micurensis (FU98), Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia lusitaniae was proven by PCR in analyzed red foxes. There is a need for continuous epidemiological monitoring of diseases caused by pathogens previously confirmed in wild canids. Also, further research is needed to reveal the role of other wild and domestic animals in the epidemiology of tick-borne zoonoses in Serbia. Divlje kanide su prepoznate kao važni domaćini rezervoari za nekoliko vektorima prenosivih zoonotskih patogena i predstavljaju dobre sentinel vrste za epidemiološki nadzor. Identifikacija domaćina rezervoara za pojedine zoonotske patogene na određenom području omogućava primenu odgovarajućih mera kako bi se sprečila pojava zoonoza kod ljudi i domaćih životinja. Cilj ove studije bio je da se predstavi uloga divljih kanida u epidemiologiji zoonoza koje se prenose vektorima u Srbiji na temelju prethodno objavljenih radova i da se dostupni podaci prodiskutuju sa epidemiološkog stanovišta. Na osnovu dosadašnjih molekularnih istraživanja u Srbiji, divlje kanide su prepoznate kao potencijalni rezervoari za nekoliko krpeljima prenosivih zoonotskih patogena. Kod šakala je potvrđeno prisustvo Anaplasma phagocytophilum, dok je prisustvo Candidatus Neoehrlichia micurensis (FU98), Borrelia burgdorferi sensu stricto, Borrelia garinii i Borrelia lusitaniae molekularnim metodama potvrđeno kod lisica. Rezultati pokazuju da su divlje kanide potencijalni domaćini rezervoari i izvor zoonotskih patogena u Srbiji i da predstavljaju dobre domaćine za više vrsta krpelja koji su potvrđeni kompetentni vektori pojedinih patogena. Potrebno je kontinuirano epidemiološko praćenje bolesti uzrokovanih patogenima čije je prisustvo prethodno potvrđeno kod divljih kanida. Takođe, potrebna su dodatna istraživanja kako bi se otkrila uloga drugih divljih i domaćih životinja u epidemiologiji zoonoza koje se prenose vektorima u našoj zemlji.

Human activities such as deforestation, urbanization, and environmental pollution lead to a reduction in the spatial boundary between wild animals, domestic animals and humans. These activities increase the risk for the emergence of pathogens from the sylvatic cycle in the population of domestic animals and humans. Foxes are recognized as potential reservoirs for a number of bacterial pathogens of medical and public health concern. The aim of the present study was to investigate the prevalence and spatial distribution of bacterial tick-borne pathogens from the Anaplasmataceae family, Borrelia burgdorferi sensu lato (s.l.), Rickettsia spp., Coxiella burnetii, Francisella tularensis, Bartonella spp., in the red fox population from Serbia and to discuss the obtained results from the epidemiological point of view. Legally hunted red foxes (Vulpes vulpes) from 14 localities in Serbia were included in the study and spleen samples from 129 animals were tested with conventional PCR assays for the presence of bacterial tick-borne pathogens. DNA of Candidatus Neoehrlichia sp. (FU98), Borrelia burgdorferi sensu stricto, Borrelia lusitaniae, and Borrelia garinii was detected in 6 (4.7%), 1 (0.8%), 2 (1.6%) and 1 (0.8%) animals, respectively. Co-infection by Candidatus Neoehrlichia sp. (FU98) and B. garinii was detected in one animal. All samples were negative for other tested bacterial tick-borne pathogens. The results of the present study indicate the potential role of foxes in natural cycles of Candidatus Neoehrlichia sp. (FU98) and causative agents of Lyme borreliosis in the investigated areas. Further research is required to elucidate the role of foxes in the epidemiology of these and other tick-borne zoonotic pathogens in the Republic of Serbia.

Spirochetes from the Borrelia burgdorferi sensu lato (s.l.). (Spirochaetales: Spirochaetaceae) species complex, including the causative agents of Lyme borreliosis, have been isolated from ticks, vertebrate reservoirs and humans. Previous analyses based on direct molecular detection in ticks indicated a considerable diversity of B. burgdorferi s.l. complex in Serbia. The present study aimed (a) to isolate borrelia strains from Serbia; (b) to determine their genotypic characteristics; and (c) to establish a collection of viable B. burgdorferi s.l. strains for further biological, ecological and genetic studies. For the present study, 231 adult Ixodes ricinus (Ixodida: Ixodidae) ticks from 16 ecologically different localities in Serbia were individually processed to cultivate B. burgdorferi s.l. This led to the isolation of 36 strains. A hbb gene quantitative real-time polymerase chain reaction (PCR) based on melting temperature determination and ospA gene sequencing were used to genotype the isolated spirochetes. The species identified based on the hbb gene real-time PCR were: Borrelia lusitaniae (44.4%), Borrelia afzelii (36.1%), Borrelia garinii (13.9%) and Borrelia valaisiana (5.6%), whereas the ospA sequence analysis revealed the occurrence of Borrelia bavariensis. This is the first report of the isolation of B. lusitaniae, B. garinii, B. bavariensis and B. valaisiana strains in Serbia.

In the present study, the effectiveness of six antimicrobial agents have been tested against 24 borrelia strains isolated from Ixodes ricinus ticks (11 Borrelia lusitaniae, eight Borrelia afzelii, three Borrelia garinii and two Borrelia valaisiana) and one B. lusitaniae strain isolated from human skin. The minimum inhibitory concentration range of antimicrobial agents was as follows: amoxicillin, 0.125–2 mg/L; doxycycline, 0.125–1 mg/L, ceftriaxone, 0.016–0.063 mg/L; cefuroxime, 0.063–1 mg/L; azithromycin, 0.0017–0.11 mg/L; amikacin 32–512 mg/L. Potentially pathogenic B. lusitaniae and B. valaisiana species were more susceptible to amoxicillin and azithromycin than pathogenic B. afzelii and B. garinii (P < 0.05); B. garinii, B. lusitaniae and B. valaisiana were more susceptible to doxycycline than B. afzelii (P < 0.05) while all species showed same susceptibility to ceftriaxone and cefuroxime (P > 0.05). This study is the first report on in vitro susceptibility of isolates from Serbia to antimicrobial agents and the first report on susceptibility of larger number of isolates of potentially pathogenic species B. lusitaniae. We showed that antimicrobial agents in vitro inhibit growth of borrelia strains very effectively, indicating the potential of their equally beneficial use in the treatment of Lyme borreliosis.

In order to determine whether conserved tick salivary protein AV422 is immunogenic, the goal of our study was to detect specific IgG response within at-risk populations. Study groups included 76 individuals, differing in occurrence of recently recorded tick bites and health status. Western blotting with recombinant (r) protein derived from Ixodes ricinus (Ir) was performed. IgG response to Borrelia/Rickettsia, as indicators of previous tick infestations, was also assessed. Additionally, a detailed in silico AV422 protein sequence analysis was performed, followed by modelling of the interactions between peptides and corresponding MHC II molecules by molecular docking. Anti-rIrAV422 seroprevalences among individuals exposed to ticks were high (62.5, 57.9 and 66.7%) and anti-Borrelia/Rickettsia seroprevalences were 54.2, 15.8 and 44.4% among individuals with/without recent tick bite and patients suspected of tick-borne disease, respectively. In silico analysis of AV422 protein sequence showed a high level of conservation across tick genera, including also the predicted antigenic determinants specific for T and B cells. Docking to the restricted MHC II molecules was performed for all predicted AV422 T cell epitopes, and the most potent (highly immunogenic) epitope determinants were suggested. The epitope prediction reveals that tick salivary protein AV422 may elicit humoral immune response in humans, which is consistent with the high anti-rIrAV422 seroprevalence in tested at-risk subjects. Tick-borne diseases are a growing public health concern worldwide, and AV422 is potentially useful in clinical practice and epidemiological studies.

Tick-borne haematozoans cause severe diseases in domestic animals, and some of them have zoonotic potential. The results of previous studies in Europe point to the important role of foxes in natural endemic cycles of several tick-borne pathogens, including protozoa. The aim of the present research was to acquire information on the prevalence and distribution of tick-borne protozoan parasites among foxes in Serbia. Legally hunted foxes from 14 localities throughout Serbia were analysed. Spleen samples were collected from 129 animals and tested for the presence of Babesia spp. and Hepatozoon spp. by PCR. In total, 79/129 (61.2%) of the tested foxes were positive for H. canis, while the presence of two Babesia species was confirmed: B. vulpes (37/129, 28.7%) and B. canis (1/129, 0.8%). Coinfection with B. vulpes and H. canis was present in 26/129 (20.2%) foxes and one animal (1/129, 0.8%) was co-infected by B. canis and H. canis. The results of this study indicate the important role of foxes in the epizootiology of B. vulpes and H. canis in the Republic of Serbia and stress the need for further research to clarify all elements of the enzootic cycle of the detected pathogens, including other reservoirs, vectors, and transmission routes.

The golden jackal (Canis aureus) is a medium-sized canid species native to Europe. This species is characterized by rapid large-scale expansion. A similar trend is also observed in Serbia, where the species is now distributed in more than a half of the territory. Although jackals prefer habitats in human-dominated landscapes, these animals have not been studied well enough from an eco-epidemiological point of view, and little is known about their potential for carrying zoonotic pathogens. In a study conducted during a three-year period (01/2010-02/2013), a total of 216 hunted or road-killed golden jackals were collected from 10 localities in Serbia. Ticks, when present, were removed, and after necropsy, spleen samples were collected from each animal. All tick and spleen samples were tested for the DNA of bacterial and protozoan tick-borne pathogens (Borrelia species, Bartonella species, Rickettsia species, Anaplasma species, Coxiella burnetii, Francisella species and Babesia species) by multiplex real-time PCR, conventional PCR and sequencing analyses. The DNA of Babesia canis was detected in nine out of 216 (4.2%) spleen samples, and two samples (0.9%) tested positive for Anaplasma phagocytophilum. In 118 ticks collected from jackals, the DNA of two Babesia species (Ba. canis and Ba. microti), three Borrelia species (Bo. garinii, Bo. valaisiana, and Bo. lusitaniae) and A. marginale was detected. From the aspect of public health surveillance, the potential role of the golden jackal in the maintenance of vector-borne zoonotic pathogens in Serbia must be considered, and further eco-epidemiological studies should be performed to determine the precise role of this animal species in zoonotic disease transmission cycles.

This study discusses a possibility of cotton fabric impregnation with pyrethrum extract as a tick repellent in supercritical carbon dioxide. A solubility of pyrethrum extract in supercritical carbon dioxide has been determined at 35 and 40 degrees C and pressures ranging from 8 to 20 MPa. The highest solubility of 48.69 kg/m(3) was obtained at temperature of 35 degrees C and pressure of 20 MPa. Density -based equations of Chrastil, Adachi-Lu and del Valle-Aguilera were employed to correlate the experimental data. Based on the solubility data, conditions for the impregnation of cotton fabric were selected. Conditions at 40 degrees C and 8 MPa provided targeted quantities of the impregnated extract of 0.5% and 1% after 1 h and 2 h of the impregnation, respectively. The presence of pyrethrins on the cotton fabric's surface was confirmed by FTIR analysis. Repellent activity of both impregnated cotton fabrics (pyrethrum extract contents of 0.5% and 1%) was proven against ticks.

Due to the recorded spreading of ticks in past years, a higher incidence of tick-borne diseases (TBDs) can be expected in the future in endemic areas, but can also pose an emerging public health concern in areas where they have not yet been recognized. Assessment of the exposure of vulnerable hosts to ticks would be a very helpful tool for TBD epidemiological studies, as well as for their proper managing. To confirm previous tick bites, the method of choice is detection of antibodies in host serum as markers developed against injected tick saliva proteins during feeding. We recently showed that the recombinant form of Ixodes ricinus AV422 saliva protein (rIrAV422) can serve for detection of markers in experimentally infested rats. Here we examine whether it can be used in the same manner in naturally exposed hosts. We chose hunting dogs as good sentinel animals. The study group consisted of 15 dogs that varied in breed, age, sex, previous tick infestation history and repellent treatment. Western blot analysis with rIrAV422 as an antigen confirmed the presence of tick bite markers in all analysed dogs. For some of the dogs, their previous tick infestation history was unclear, which emphasizes the usefulness of rIrAV422 for revealing it. Since hunting dogs are naturally infested with different ticks, the potential of rIrAV422 in assessment of general exposure to ticks is highlighted. Use of rIrAV422 can also be helpful in veterinary practice and research as a tool for validation of the efficiency of tick repellent products.

Tick bites often go unnoticed, so specific reliable tests are needed to confirm them for prompt diagnosis and treatment of tick-borne diseases. One of the promising candidates for developing such a test is AV422, a tick saliva protein that has been conserved across tick genera. In this study, we demonstrate the potential of the AV422 homologue from Ixodes ricinus to be used for tick bite detection for both Prostriata and Metastriata. We expressed recombinant (r) I. ricinus (Ir) AV422 in E. coli and subjected it to Western blot analysis using rat antibodies to saliva proteins of both I. ricinus (Prostriata) and Dermacentor reticulatus (Metastriata) larvae. Our data demonstrate that rIrAV422 specifically bound to antibodies from sera of rats used for both I. ricinus and D. reticulatus larvae feeding, but not to antibodies from control serum, emphasizing its specificity since tick bites were the sole cause of sera reactivity.

Specimens of the endangered species Spermophilus citellus (European ground squirrel) from populations inhabiting 12 localities in Vojvodina (Serbia) were inspected for the presence of attached hard ticks. A total of 1009 ticks were found on 151 infested animals. With respect to the level of infestation, over 60% of the squirrels were found with five or less ticks attached, while the distribution of the number of animals with a higher level of infestation tended to follow the 80/20 rule. A notably higher level of infestation was detected in males as compared to females. Nymphs were the predominant developmental stage of ticks parasitizing the inspected European ground squirrel specimens. The majority of removed ticks belonged to the nidicolous species Ixodes laguri (79%), while the remaining ticks were identified as Haemaphysalis concinna. In seeking molecular confirmation of morphological identification of Ixodes specimens, COI gene sequences of I. laguri were obtained and deposited in GenBank.

Ticks parasitizing bats have been largely understudied, especially in the central part of the Balkan Peninsula, where the last data from the field research date from almost 25 years ago. Bats are hosts to a large number of ectoparasites, including ticks, which can act as vectors of zoonotic agents. For this reason, it is important to identify the distribution of ticks and their relationship to different hosts, including wild animals, bats in particular. The present research was conducted at 16 localities throughout Serbia, Montenegro, Bosnia and Herzegovina, and Former Yugoslav Republic of Macedonia (FYROM). We examined 475 individuals of bats belonging to 13 species. A total of three tick species were identified, I. simplex being the most numerous and widespread, followed by I. vespertilionis and A. vespertilionis. To the best of our knowledge, the presented data include the first records of I. simplex in Serbia and Montenegro, I. vespertilionis for Montenegro and A. vespertilionis in FYROM. Also, we identify a new possible host/parasite association between I. simplex and Rhinolophus euryale.

The golden jackalCanis aureusoccurs in south-eastern Europe, Asia, the Middle East, the Caucasus, and Africa. In Serbia, jackals neared extinction; however, during the last 30 years, the species started to spread quickly and to increase in number. Few studies in the past have revealed their potential role as carriers of zoonotic diseases. Animal samples were collected over a three-year period (01/2010–02/2013) from 12 sites all over Serbia. Of the tissue samples collected, spleen was chosen as the tissue to proceed; all samples were tested forLeishmaniaspecies andBrucellaspecies by real-time PCR. Of the 216 samples collected, 15 (6.9%) were positive forLeishmaniaspecies, while four (1.9%) were positive forB. canis. The potential epidemiologic role of the golden jackal in carrying and dispersing zoonotic diseases in Serbia should be taken under consideration when applying surveillance monitoring schemes.

The golden jackal Canis aureus occurs in south-eastern Europe, Asia, the Middle East, the Caucasus and Africa as well as in countries of the European continent, including Serbia. The scope of the current survey was to study the presence of two zoonotic agents, Leishmania species (vector transmitted) and Brucella species, in the jackal population in Serbia. Animal samples were collected over a three-year period (01/2010 - 02/2013) from 48 localities corresponding to 12 regions of Serbia. Sampling took place from dead animals brought at the laboratory of Department for Animal Ecology and Geography, Faculty of Biology, University of Belgrade, by hunters. Data regarding location were recorded. At necropsy, samples from different tissues were collected (spleen, liver, hart); spleen was chosen as the tissue to work with. Samples were homogenised with micropastles (Eppendorf). DNA extraction took place from a small portion of smashed tissue using the Gene Jet Genomic DNA Purification Kit, (Fermentas, Thermo Scientific). For the detection of Brucella species, a multiplex Real-time PCR protocol was used, targeting the bcsp31, alkB and BMEI1162 genes of Brucella species, B. abortus and B. melitensis; B. canis was detected using a PCR protocol targeting the omp2B gene. For the detection of Leishmania species a Real-time PCR protocol targeting the SSU rRNA gene was used. A total of 216 spleen samples were tested. Most samples (196/90.7%) were collected during the hunting period (November-February). Of the samples tested, 108 animals were male and 108 were female. Fifteen (6.9%) were positive for Leishmania species, while four (1.9%) were positive for B. canis. Jackals are known omnivores and scavengers and they usually prey on small mammals, on vegetables, fruit and garbage. Their ability to adapt to novel habitats both on rural and urban areas allows them to come into contact with animals that live in close proximity to humans, such as dogs, hence their ability to host a number of zoonotic, including vector-borne, pathogens. An increase in jackal population has lately been observed in Serbia, while complaints about damages that jackals may cause to livestock mean that their wildlife cycle may well interfere with areas of natural human activity. Although further studies are required to discern the potential epidemiologic role of the golden jackal in spreading and transmitting the above studied pathogens, local awareness of residents, veterinarians, and health professionals would prove of great importance in order to efficiently monitor these and other zoonotic diseases.

The geographic distribution of Borrelia burgdorferi sensu lato species in Europe shows very dynamic spatial and temporal variations. Since different Borrelia species usually correlate with different clinical manifestation of Lyme borreliosis, knowledge of the geographic distribution of the pathogen is very important for understanding the ecology and epidemiology of the disease. Our previous studies, based on direct molecular methods, revealed high diversity of species of the B. burgdorferi s.l. complex and unexpected high prevalence of B. lusitaniae in Ixodes ricinus ticks from Serbia. The aim of the present study was further isolation and typization of viable B. burgdorferi s.l. strains from vectors in Serbia as a basis for studies of biological, genetic and ecological variations. A total of 248 adult I. ricinus ticks collected from 24 localities were processed for cultivation. Prior to cultivation, all ticks were disinfected, then triturated in BSK H medium. The culture tubes were incubated at 33°C for 3 months. Cultures were periodically examined by dark field microscopy. For confirmation of isolated strains, "seminested" PCR for the flaB gene was performed. Determination of B. burgdorferi s.l. species was carried out by the RFLP technique, using restriction enzymes MseI and DraI on the previously amplified 5-23S rDNA intergenic spacer (Postic et al., 1994). For Borrelia identification by RT-PCR targeting, the hbb gene protocol of Portnoi et al. (2006) was followed. Sequencing of the 5-23S rDNA intergenic spacer and flaB gene was performed for phylogenetic analysis. Thirty-four spirochete cultures were isolated and subjected to further genotyping analyses. According to the RFLP patterns of the 5S-23S rDNA intergenic spacer, specimens were determined as: B. lusitaniae, B. afzelii, B. burgdorferi s.s., B. garinii and B. valaisiana. For all strains identified as B. lusitaniae, B. garinii, B. afzelii and B. valaisiana according to the MseI-RFLP method, results of RT-PCR were in absolute agreement. However, Tm values for all strains identified as B. burgdorferi s.s. according to RFLP patterns were in the range for B. lusitaniae strains (64.17 to 64.58 °C). Thus, it was impossible to distinguish B. burgdorferi s.s. and B. lusitaniae strains by this method. Sequencing analysis for strains identified as B. lusitaniae, B. garinii, B. afzelii and B. valaisiana was in agreement with MseI-RFLP and RT-PCR results. In cases of uncoordinated MseI-RFLP and RT-PCR results, sequencing analysis confirmed unclear strains as B. lusitaniae. The results of this study showed B. lusitaniae to be the species with highest prevalence in I. ricinus ticks from Serbia.