Search

Background: Borrelia lusitaniae is a species within the complex Borrelia burgdorferi sensu lato, associated with lizards as reservoirs and Ixodes ricinus as its main vector. Borrelia lusitaniae is predominantly distributed in Central and Southeast Europe, and in countries of the Mediterranean basin, such as Portugal, Morocco, Tunisia, and Italy where this spirochete appears to infect vector ticks more frequently than other genospecies. Evolution of this zoonotic tick-borne microparasite is shaped by different environmental factors. Comprehensive phylogenetic analysis may give insight into how B. lusitaniae spreads to new geographic locations. Aim: We applied Bayesian statistical methods to B. lusitaniae multilocus sequence typing (MLST) data to study the migration routes of B. lusitaniae and its potential for further spread. Results: The discrete phylogeographic analysis placed origins of B. lusitaniae in Southeast Europe and identified at least two introductions of B. lusitaniae from Europe to North Africa. Estimated effective reproductive potential (Re), as a key indicator for a pathogen spread, suggested potential for further spread. Conclusion: The results of this study can provide beneficial information about the potential for further spread of B. lusitaniae in Europe and North Africa and estimation of necessity for the development of strategies to monitor and control Lyme borreliosis.

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.

Introduction: Zoonotic tick-borne diseases (TBDs) represent an increasing threat in urban areas, including city parks and green recreational areas. Borrelia miyamotoi is pathogenic to humans but there is still little information about its circulation in nature and potential local impact on human health, while tick-borne encephalitis virus (TBEV) is endemic in many European countries and sporadic autochthonous cases in humans have been reported in Serbia. The objective of our study was to determine the prevalence of two emerging zoonotic tick-borne pathogens (TBPs) − B. miyamotoi and TBEV in urban areas in the city of Belgrade (24 localities), four localities in Eastern Serbia, one in the province of the Vojvodina. Methods: At 29 localities, a total of 480 ticks were collected from March to June 2021, and identified to the species level by using morphological keys. DNA and RNA were extracted from individual tick samples, while for molecular detection, probe-based qPCR and nested PCR were applied. Results: Among 480 ticks, 445 were identified as Ixodes ricinus (213 males, 176 females, 56 nymphs), 30 as Dermacentor reticulatus (18 females, 12 males), three Dermacentor marginatus females, one Haemaphysalis concinna female, and one Haemaphysalis punctata male. Using probe-based qPCR, the prevalence of B. miyamotoi in ticks was 1.04% (DNA was detected in five I. ricinus, two females and two males collected from three Belgrade localities and one male from Eastern Serbia), while sequencing was successful in samples of two I. ricinus females collected from two Belgrade localities and one male from Eastern Serbia. TBEV RNA was not detected in any sample. Conclusions: This is the first report on the presence of the emerging zoonotic pathogen B. miyamotoi in I. ricinus ticks in urban green areas in Serbia, indicating the risk of B. miyamotoi disease. Risk areas within cities should be identified and knowledge regarding TBPs and TBDs among the general population in urban areas s

Kompleks Borrelia burgdorferi sensu lato trenutno obuhvata dvadest i jednu opisanu vrstu borelija a karakteriše ih značajna varijabilnost u pogledu geografske distribucije kao i specifičnosti na relaciji domaćini rezervoari- kompetentni vektor. Poznato je da samo određeni sojevi vrsta borelija izazivaju bolest kod ljudi. U Severnoj Americi, lajmsku boreliozu uzrokuje samo jedna vrsta borelija-Borrelia burgdorferi sensu stricto (s.s.), dok u Evropi infekciju kod ljudi može uzrokovati nekoliko različitih vrsta borelija-Borrelia afzelii, Borrelia garinii, Borrelia bavariensis, Borrelia spielmanii i Borrelia burgdorferi s.s. Vrste Borrelia valaisiana, Borrelia lusitaniae i Borrelia bissettii identifikovane su samo u pojedinačnim slučajevima i prepoznate su kao potencijalni patogeni. Različite vrste borelija povezane su sa različitim kliničkim manifestacijama lajmske borelioze; B. afzelii obično ostaje lokalizovana u koži, B. garinii i B. bavariensis se obično povezuju s poremećajima nervnog sistema, dok se B. burgdorferi s.s. često povezuje sa razvojem artritisa. Iako postoje registrovani slučajevi lajmske borelioze u Srbiji, ne postoje podaci o tome koje vrste borelija izazivaju određene kliničke manifestacije lajmske borelioze. Region Srbije karakteriše visoka rasprostranjenost i raznovrsnost vrsta borelija u krpeljima i njihovim domaćinima. Istraživanja prisustva borelija u krpeljima kao i mikrobiološka izolacija sojeva borelija iz krpelja u Srbiji, ukazala su na dominaciju vrste B. lusitaniae, slede vrste B. afzelii, B. bavariensis, B. garinii, B. valaisiana i B. burgdorferi s.s. Takođe, B. lusitaniae je detektovana u krpeljima sakupljenim sa guštera i u uzorcima slezine lisica, dok je B. valaisiana detektovana u krpeljima sakupljenim sa šakala i ptica. Ispitivanje in vitro osetljivosti lokalnih sojeva borelija (izolovanih iz krpelja vrste Ixodes ricinus sakupljenih sa vegetacije) na ljudski serum, pokazalo je da su sojevi B. valaisiana otporni na komplement, dok su, The twenty-one described Borrelia species with considerable variability in host and vector associations, and geographical distribution have been classified to the Borrelia burgdorferi sensu lato complex. Only a subset of Borrelia species is known to cause human illness. In North America, Lyme borreliosis (LB) is predominantly caused by the Borrelia burgdorferi sensu stricto (s.s.), while in Europe, the overall diversity of Borrelia species is higher and LB can be caused by Borrelia afzelii, Borrelia garinii, Borrelia bavariensis, Borrelia spielmanii, and Borrelia burgdorferi s.s., while Borrelia valaisiana, Borrelia lusitaniae and Borrelia bissettii have been identified in single cases only and are recognized as potential pathogens. Different Borrelia species are involved in distinct clinical manifestations of LB; B. afzelii usually remains localized in the skin, B. garinii and B. bavariensis are usually associated with nervous system disorders, while B. burgdorferi s.s. is commonly associated with development of the arthritis. Although there are registered cases of LB in Serbia, there is no data on which Borrelia species cause certain clinical manifestations of LB. Region of Serbia is characterized by high prevalence and diversity of Borrelia species in ticks and their vertebrate hosts. The studies on the presence and isolation of Borrelia from ticks from Serbia pointed to the domination of B. lusitaniae followed by B. afzelii, B. bavariensis, B. garinii, B. valaisiana and B. burgdorferi s.s. Also, B. lusitaniae was detected in ticks collected from lizards and in spleen samples collected from red foxes, while B. valaisiana was detected in ticks collected from golden jackals and birds. In vitro susceptibility of local Borrelia strains isolated from ticks to human complement, showed B. valaisiana strains resisted complement-mediated killing, while certain B. lusitaniae strains (in the presence of complement) were equally motile as the only existing one B. lusitaniae

Tropical Diseases of the University Clinical Centre of Serbia with a clinical presentation of disseminated erythema migrans. The patient and the mother could not recall if there had been a tick bite. A sample of blood was taken, and antibiotic therapy with amoxicillin was started immediately. Human serum sample was checked for the presence of IgM antibody against Borrelia burgdorferi sensu lato by commercial ELISA test and the sample was positive for IgM. Blood was collected into the sterile K2EDTA tube, immediately transported to the Laboratory at the Institute for Medical Research, National Institute of the Republic of Serbia, University of Belgrade, and centrifuged twice at 2200 rpm for 17 min at room temperature. After centrifugation, one part of the serum was served for DNA extraction using ammonium hydroxide, ethanol, and sodium acetate while the sediment was inoculated into a 6 mL tube with Barbour-Stoenner-Kelly-H (BSK-H) medium under aseptic conditions and incubated as 33°C. After 16 days of incubation, viable, motile, and spiral-shaped microorganisms were observed in the initial BSK-H culture under dark-field microscopy, and incubation was prolonged for 29 days. DNA from the culture was extracted using centrifugation, dissolving the sediment in the water, and heating at 95°C for 10 minutes. After extracting DNA from the human serum and the culture, rrf-rrl rDNA intergenic spacer and flagellin gene (flaB) were amplified by conventional PCR, and sequencing of obtained PCR products was performed commercially (Macrogen, Amsterdam, the Netherlands). After analysis of the sequences obtained, Borrelia lusitaniae was confirmed in human serum and culture. This is the first isolate of B. lusitaniae from a human blood sample that confirms that B. lusitaniae can disseminate via the hematogenous route.

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.

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.

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.

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.

Assessment of exposure to ticks is a significant data in epidemiological studies of tick-borne diseases, and confirmation of tick bite is important for their diagnosis. In this study, three saliva proteins, PA107, AV422 and calreticulin (CAL), of Ixodes ricinus tick, medically most important species in Europe, were examined in the context of their use for detection of specific antibodies in sera of hosts, as bite markers. Sequence variability analysis revealed high conservativity of all three proteins, with PA107 as the most conservative. Since CAL is ubiquitous in eukaryotes, the further focus of this study was on PA107 and AV422, as proteins that are specific for ticks. Homologues of these proteins from Ixodes ricinus were analyzed for the first time. Reactivity of sera of experimental animals, rats, which were infested separately with Ixodes ricinus or Dermacentor reticulatus larvae, with recombinant forms of IrPA107 and IrAV422 were examined. Specific antibodies were detected in sera of all infested rats using rIrAV422, which implied its usage for detection of bites of tick species that belong to different groups of ixodid ticks, with high sensitivity and specificity. Reactivity with IrPA107 was significantly lower. Hunting dogs, as good sentinels, were used for assessment of rIrAV422 usage for detection of markers in sera of hosts which are exposed to ticks in natural environment. Obtained seroreactivity in all dogs implies broad applicability of this saliva protein, in terms of assessment of exposure to different tick species and risk of infection with tick-borne pathogens., Procena izloženosti krpeljima je značajna u epidemiološkim studijama oboljenja čije uzročnike prenose krpelji, a potvrda uboda krpelja je važna za njihovu dijagnostiku. U ovom radu su ispitana tri proteina pljuvačke, PA107, AV422 i kalretikulin (CAL), krpelja Ixodes ricinus, medicinski najznačajnije vrste u Evropi, u kontekstu upotrebe za detekciju specifičnih antitela u serumu domaćina, kao markera uboda. Analiza varijabilnosti njihovih sekvenci je ukazala na visoku konzerviranost sva tri proteina, pri čemu se PA107 pokazao kao najkonzerviraniji. S obzirom na ubikvitarnu zastupljenost CAL kod eukariota, dalji fokus u ovoj studiji je bio na PA107 i AV422, kao proteinima koji su specifični za krpelje i čiji homolozi su prvi put analizirani kod vrste Ixodes ricinus. Ispitivana je reaktivnost seruma eksperimentalnih životinja, pacova, na kojima su hranjene larve Ixodes ricinus ili Dermacentor reticulatus, sa rekombinantnim formama IrPA107 i IrAV422. Korišćenjem rIrAV422, specifična antitela su detektovana kod svih infestiranih pacova, što ukazuje da se rIrAV422 može upotrebiti za detekciju markera uboda različitih grupa tvrdih krpelja uz visoku senzitivnost i specifičnost. Reaktivnost u slučaju IrPA107 je bila značajno manja. Za ispitivanje upotrebe rIrAV422 u detekciji markera uboda u serumima domaćina izloženih krpeljima u prirodnom okruženju, korišćeni su lovački psi kao adekvatna indikatorska grupa. Dobijena seroreaktivnost kod svih analiziranih pasa u studiji ukazuje na širok potencijal primene ovog proteina pljuvačke u proceni izloženosti različitim vrstama krpelja i rizika od infekcije patogenima koje oni prenose.

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.

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%). Co-infection 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.

In order to evaluate the prevalence rate of Babesia spp. in ticks collected from vegetation at seven localities in northern Serbia, tick samples were subjected to molecular analysis. A total of 132 unfed adult ticks of five different species (Dermacentor marginatus, Dermacentor reticulatus, Ixodes ricinus, Haemaphysalis concinna and Haemaphysalis punctata), were examined by PCR for the presence of Babesia spp. Out of the analyzed ticks, 10.61% (14/132) were positive for babesial DNA. The presence of babesiae was found at the localities Pančevački Rit, Titov Gaj, Makiš, PKB and Kljajićevo. Prevalence in D. reticulatus ticks was 21.57% (11/51) and in H. concinna ticks, 8.57% (3/35). Sequencing and phylogenetic analysis showed a clustering of the obtained sequences with those of B. canis from the GenBank database. These results add to the knowledge of the distribution of babesial pathogens and their vectors in Serbia.

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.

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.

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.