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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.

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.

The Ixodes ricinus tick is common in the central part of the Balkan Peninsula. It is a vector of pathogenic agents causing diseases in humans and animals. Little is known about the genetic structure of I. ricinus in this region. We have investigated intraspecific variability of the COI gene among I. ricinus ticks collected from different regions of Serbia, and the correlation between the various types of habitat and genetic variability of ticks. The obtained COI gene sequences are the first barcoding sequences of I. ricinus ticks collected at localities in Serbia. Intraspecific variability of these COI gene sequences was very low, and there was no correlation between the various types of habitat and genetic variability of ticks. Samples from isolated localities (canyon/gorge) showed no genetic differentiations from the majority of samples from open areas.

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.

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.

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.

Previous results indicate that glycerol-3-phosphate dehydrogenase variability represents the adaptation of Ixodes ricinus L. (Acari: Ixodidae) to fluctuations of environmental conditions, particularly to temperature. Analysis of crucial polymorphisms in I. ricinus Gpdh gene was done by the restriction method, and three different haplotypes were obtained (GPDH(441) 1, GPDH(441) 2, and GPDH(441) 3), corresponding to GPDH alleles detected by allozyme electrophoresis. Differences in GPDH(441) haplotype and genotype frequencies were found between samples from open and forest habitats. Significant seasonal variations of GPDH(441) haplotype and genotype frequencies were detected in samples from the open habitats. No seasonal variations were observed at forest localities, probably because of the less pronounced amplitude of environmental factors. The possible role of host availability was discussed as an important factor that affects seasonal dynamics and genetic composition of tick populations.