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The paper reports the prevalence and geographical distribution of Borrelia burgdorferi sensu lato (s.l.) and its genospecies in 12,221 questing Ixodes ricinus ticks collected at 183 locations from all the 41 counties of Romania. The unfed ticks were examined for the presence of B. burgdorferi s.l. by PCR targeting the intergenic spacer 5S-23S. Reverse line blot hybridization (RLB) and restriction fragment length polymorphism (RFLP) analysis were performed for identification of B. burgdorferi genospecies. The overall prevalence of infection was 1.4%, with an average local prevalence between 0.75% and 18.8%. B. burgdorferi s.l. was found in ticks of 55 of the 183 localities. The overall prevalence B. burgdorferi s.l. in ticks in the infected localities was 3.8%. The total infection prevalence was higher in female ticks than in other developmental stages. Three Borrelia genospecies were detected. The most widely distributed genospecies was B. afzelii, followed by B. garinii and B. burgdorferi sensu stricto (s.s.). The study is the first countrywide study and the first report of B. burgdorferi s.s. in Romania. The distribution maps show that higher prevalences were recorded in hilly areas, but Lyme borreliosis spirochetes were also present in forested lowlands, albeit with a lower prevalence.

In 2010 and 2011, questing ticks were collected from 188 forested locations in all the 41 counties of Romania using the dragging method. The total of 13,771 ticks collected belonged to eleven species: Ixodes ricinus (86.9 %), Dermacentor marginatus (9.5 %), Haemaphysalis punctata (2.6 %), H. concinna (0.6 %), H. sulcata (0.3 %), H. parva (0.1 %), Hyalomma marginatum (0.02 %), D. reticulatus (0.02 %), I. crenulatus (0.007 %), I. hexagonus (0.007 %) and I. laguri (0.007 %). Ixodes ricinus was present in 97.7 % (n = 180) of locations, occurring exclusively in 41.7 % of the locations, whereas it was the dominant species in 38.8 % of the other locations, accounting for over 70 % of the total tick community. The following most common questing ticks were D. marginatus, H. punctata and H. concinna. Ixodes ricinus co-occurred with one, two or three sympatric species. The occurrence of D. reticulatus in forested habitats from Romania was found to be accidental.

Granulocytic anaplasmosis is a common vector-borne disease of humans and animals with natural transmission cycle that involves tick vectors, among which Ixodes ricinus is the most important. The present paper reports the prevalence and geographical distribution of A. phagocytophilum in 10,438 questing Ixodes ricinus ticks collected at 113 locations from 40 counties of Romania. The unfed ticks were examined for the presence of A. phagocytophilum by PCR targeting a portion of ankA gene. The overall prevalence of infection was 3.42%, with local prevalences ranging between 0.29% and 22.45%, with an average prevalence of 5.39% in the infected localities. The infection with A. phagocytophilum was detected in 72 out of 113 localities and in 34 out of 40 counties. The highest prevalence was recorded in females followed by males and nymphs. The results and the distribution model have shown a large distribution of A. phagocytophilum, covering Romania's entire territory. This study is the first large scale survey of the presence of A. phagocytophilum in questing I. ricinus ticks from Romania.

Migratory birds play important roles as distributors of ticks within and between continents. In the Old World, the most important migratory route of birds links Asia, Europe and Africa. During their migration, birds use various stopover sites, where they feed and rest and where ticks may attach or detach, creating new natural foci for vector-borne diseases. Danube Delta is one of the most important migration hotspots and so far no studies were focused on ticks of migratory birds herein. The aim of the present study was to assess the species diversity and seasonal dynamics of ticks parasitizing migratory birds in Danube Delta Biosphere Reserve. Migratory birds were trapped on Grindul Lupilor (44°41′N; 28°56′E) using mist nets during 4 migratory seasons (2 spring and 2 autumn) in 2011 and 2012. From each bird, all the ticks were collected and identified based on morphological features. Epidemiological parameters (prevalence, mean abundance, mean intensity) were calculated and all data were analysed statistically based on the season (spring and autumn), regional status of birds (migrants and breeding) and foraging behaviour (ground feeders, reed-bed feeders, foliage feeders). A total of 1434 birds (46 species) were captured. Ticks were found on 94 birds (10 species). Significantly more migratory birds hosted ticks, compared to resident birds. The 400 collected ticks belonged to four species: Ixodes ricinus (92.25%), I. arboricola (6.25%), I. redikorzevi (1.00%) and Haemaphysalis punctata (0.50%). A higher prevalence was found for I. ricinus in spring, with higher prevalence of nymphs in this season, while larvae occurred with the same prevalence in both seasons. Larval intensity was higher during spring and nymphs were more abundant during autumn. The seasonal differences in our study may be related not to the local seasonal dynamics of ticks, but on the seasonal dynamics at the site of migration initiation.

Testudo graeca tortoises are distributed in the south-eastern part (Dobrogea region) of Romania. T. graeca is a potential host for the three-host ticks, Hyalomma aegyptium. H. aegyptium ticks are important from epidemiological point of view as they constitute potential reservoirs for numerous zoonotic bacterial pathogens (Anaplasma phagocytophilum, Ehrlichia canis, Coxiella burnetii). However, H. aegyptium was reported to host less studied bacteria, non-Lyme members of genus Borrelia. Despite its relatively wide distribution range, the extent of co-distribution of ticks with these bacteria was not investigated in detail. The aim of the present study was to evaluate H. aegyptium engorged ticks collected from tortoises in south-eastern Romania for the presence of non-Lyme Borrelia. Between 2008 and 2013, 448 H. aegyptium ticks were collected from 45 T graeca tortoises located in Dobrogea region in Romania. DNA extraction was performed individually from each tick using a commercial kit. For the total 78 (17.4%) Borrelia spp. positive ticks, PCR analysis targeting the intergenic spacer 5S-23S region, glpQ, respectively gyrB genes, and further sequencing was performed for the further identification. Sequences of gyrB and glpQ genes showed 99%-100% similarities with reptile-associated Borrelia turcica. The most frequently infected stages were males (10.7% of the total males examined or 61.5% from the total infected ticks) followed by females (5.36% of the total females examined or 31% from the total infected ticks) and nymphs (1.34% of the total nymphs examined or 7.7% from the total infected ticks). This is the first report of Borrelia turcica in Romania. This research was supported by grant CNCSIS IDEI PCCE 7/2010 and IDEI PCE 236/2011.