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Red foxes, Vulpes vulpes are among the most widely spread carnivores in the world, invading also urban areas and are often parasitized by various ticks and directly exposed to several vector-borne pathogens, including the commonly present "Theileria annae". Considering the paucity of data on the possible vectors of this pathogen and the presence of the infection in various locations across the globe, the aim of our study was to understand the potential role of various environmental factors on the distribution of "T. annae" in red foxes from a well-defined region within the Carpathians, Romania. Between July 2016 and April 2017, a total of 347 blood samples originating from red foxes from 13 counties were tested using a PCR specifically designed for "T. annae". In order to assess the potential distribution of "T. annae" based on niche modelling, we used presence-only data and 15 ecological variables. The probability of presence models was built using MaxEnt software. Of all sampled foxes, 20.1% (66 unique locations in 8 counties) were positive for "T. annae" DNA. There was no significant difference between the prevalence in males and females, nor between juveniles and adults. The sequences were all identical to each other and showed 100% identity to other sequences deposited in GenBank. The highest contribution to the spatial model was represented by the agricultural land coverage. This is the first study to demonstrate the presence of "T. annae" in foxes in Romania and the first spatial analysis for "T. annae" highlighting the importance of the environmental factors on its distribution., (Copyright © 2018. Published by Elsevier GmbH.)

Crows (Corvidae) are common city dwellers worldwide and are increasingly important subjects of epidemiology studies. Although their importance as hosts and transmitters of a number of zoonotic parasites and pathogens is well known, there are no studies on their importance as tick hosts. After mosquitoes, ticks are the most important vectors of zoonotic pathogens, especially for those causing emerging zoonotic diseases. Pathogenic bacteria, especially Borrelia spp., Rickettsia spp., and Anaplasma spp., vectored by ticks, are the cause for most vector-borne diseases in Europe. Here we report on ticks and tick-borne pathogens harbored by urban breeding crows. A total of 36 birds (33.33%, n = 108) hosted ticks, with 91 individual ticks belonging to 6 species (Haemaphysalis concinna, Haemaphysalis parva, Haemaphysalis punctata, Hyalomma marginatum, Ixodes arboricola, and Ixodes ricinus). Rickettsia spp. DNA was found in 6.6% of ticks and 1.9% of bird tissues, whereas Anaplasma phagocytophilum was found in 5.9% of ticks and 0.9% of birds. Two rickettsial genospecies were located, Rickettsia helvetica and Rickettsia monacensis. This is the first study to determine such a diverse tick spectrum feeding on urban corvids, while highlighting their importance as tick hosts and raising concerns about their potential risk to human health.

Background: Birds are important hosts and dispersers of parasitic arthropods and vector-borne zoonotic pathogens. Particularly migratory species may carry these parasites over long distances in short time periods. Migratory hotspots present ideal conditions to get a snapshot of parasite and pathogen diversity of birds migrating between continents. The aim of this study was to investigate the presence and diversity of Rickettsia spp. in ticks collected from birds at a migratory hot-spot in the Danube Delta, Romania, eastern Europe., Methods: DNA was extracted from ticks that were collected from migratory birds in the Danube Delta during migratory seasons in 2011-2012. Two 360 bp  fragments of the 16S ribosomal RNA gene and a 381 bp  fragment Gene gltA were PCR amplified and analyzed by sequence analysis (performed at Macrogen Europe, Amsterdam, The Netherlands). Nucleotide sequences were compared to reference sequences available in the GenBank database, using Basic Local Alignment Search Tool., Results: Four hundred ticks of four different species were found on 11 bird species. The prevalence of Rickettsia spp. infection was 14 % (56/400, CI: 11.7-29.1), with significantly more nymphs hosting rickettsial infection compared to larvae (48 vs 7; P < 0.001). Significantly more ticks in nymphal stage were hosting Rickettsia spp. infection in spring, than in autumn. Four different genospecies were found: R. monacensis (29 ticks), R. helvetica (13), R. massiliae (3) and R. slovaca (2). The seasonal distribution of different Rickettsia spp. was heterogeneous; with most of the R. monacensis-infected ticks were found in spring, while more R. helvetica were found in autumn than spring. R. massiliae was found only in autumn and R. slovaca was found only in spring., Conclusion: This study has shown that birds migrating through eastern Europe may carry ticks infected with a high diversity of rickettsial pathogens, with four Rickettsia spp. recorded. Migratory direction was important for pathogen burden, with seasonal differences in the occurrence of individual Rickettsia species. Here we report the first individual records of different Rickettsia spp. in H. concinna (R. monacensis), I. arboricola (R. helvetica, R. massiliae) and I. redikorzevi (R. helvetica) and also the first geographical record of occurrence of R. massiliae in Romania, representing the easternmost observation on the continent.

Background: Red foxes (Vulpes vulpes) are one of the most widespread wild carnivores in the world, being recognized to harbor and transmit a wide range of vector-borne diseases. Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato are zoonotic tick-borne pathogens causing emerging diseases. Wild animals play an essential role in the transmission of diseases and pathogens maintenance in nature. Epidemiological studies regarding the prevalence of tick-borne pathogens in red foxes are of public health importance, as they may successfully act as a pathogen transmission interface between wildlife, domestic animals and humans., Findings: This study included 14 counties from Romania. A total number of 353 red foxes (Vulpes vulpes) were examined. Heart tissue samples were collected during necropsy and stored at -20 °C. Genomic DNA extraction was performed and all samples were examined by polymerase chain reaction (PCR). Specific primers for A. phagocytophilum, A. platys, E. canis and Borrelia burgdorferi s.l. were used. Sequence analysis was performed (Macrogen Europe, Amsterdam) and obtained sequences are available at GenBank™. Out of the 353 samples, 9 (2.55 %; 95 % CI: 1.25-4.96 %) were positive for A. phagocytophilum. Positive animals originated from 5 counties. In total, 5 out of 353 heart tissue samples (1.42 %; 95 % CI: 0.52-3.47 %) collected from red foxes were positive for B. burgdorferi s.l. Red foxes originated from 4 counties. None of the samples were positive for A. platys or E. canis. No co-infection with A. phagocytophilum and B. burgdorferi s.l. was found., Conclusion: This first report of A. phagocytophilum and B. burgdorferi s.l. in red foxes from Romania suggests a limited role of foxes in the maintenance of the two related pathogens, but may represent a potential risk from a public health perspective.

Background: Ticks are among the most important vectors of zoonotic diseases in temperate regions of Europe, with widespread distribution and high densities, posing an important medical risk. Most ticks feed on a variety of progressively larger hosts, with a large number of small mammal species typically harbouring primarily the immature stages. However, there are certain Ixodidae that characteristically attack micromammals also during their adult stage. Rodents are widespread hosts of ticks, important vectors and competent reservoirs of tick-borne pathogens. Micromammal-tick associations have been poorly studied in Romania, and our manuscript shows the results of a large scale study on tick infestation epidemiology in rodents from Romania., Methods: Rodents were caught using snap-traps in a variety of habitats in Romania, between May 2010 and November 2011. Ticks were individually collected from these rodents and identified to species and development stage. Frequency, mean intensity, prevalence and its 95% confidence intervals were calculated using the EpiInfo 2000 software. A p value of <0.05 was considered statistically significant., Results: We examined 423 rodents (12 species) collected from six counties in Romania for the presence of ticks. Each collected tick was identified to species level and the following epidemiological parameters were calculated: prevalence, mean intensity and mean abundance. The total number of ticks collected from rodents was 483, with eight species identified: Ixodes ricinus, I. redikorzevi, I. apronophorus, I. trianguliceps, I. laguri, Dermacentor marginatus, Rhipicephalus sanguineus and Haemaphysalis sulcata. The overall prevalence of tick infestation was 29.55%, with a mean intensity of 3.86 and a mean abundance of 1.14. Only two polyspecific infestations were found: I. ricinus + I. redikorzevi and I. ricinus + D. marginatus., Conclusions: Our study showed a relatively high diversity of ticks parasitizing rodents in Romania. The most common tick in rodents was I. ricinus, followed by I. redikorzevi. Certain rodents seem to host a significantly higher number of tick species than others, the most important within this view being Apodemus flavicollis and Microtus arvalis. The same applies for the overall prevalence of tick parasitism, with some species more commonly infected (M. arvalis, A. uralensis, A. flavicollis and M. glareolus) than others. Two rodent species (Mus musculus, Rattus norvegicus) did not harbour ticks at all. Based on our results we may assert that rodents generally can act as good indicators for assessing the distribution of certain tick species.

Simple Summary: Ticks are important parasites that feed on the blood of various host species, representing the most important arthropods transmitting diseases in Europe. Continuous changes in both tick distribution and abundance are related to multiple factors, including climate change. These changes have strong implications for both animal and human health; therefore, continuous surveillance of tickborne diseases is required for an appropriate evaluation of the potential risks faced by animals and humans in a given area. The spotted fever group Rickettsia comprises a large number of zoonotic agents with an increasing importance recognized in the last 30 years. The aim of this study was to evaluate these bacteria in ticks in Romania. Five Rickettsia species were identified in different tick species, with new pathogen–tick associations reported. Rickettsia hoogstraalii, one member of this group, was detected for the first time in Romania and in Rhipicephalus rossicus ticks. This species was first described in 2006 in Croatia, and its pathogenicity is not well known. In addition, the detection of R. raoultii and R. monacensis in unfed larvae of Haemaphysalis punctata reinforce the hypothesis of transmission of Rickettsia from female ticks to larvae; therefore the bite of larvae could pose a health risk. Tickborne bacterial pathogens have been described worldwide as risk factors for both animal and human health. Spotted fevers caused by Rickettsiae may cause non-specific symptoms, which make clinical diagnosis difficult. The aim of the current study was to evaluate and review the diversity of SFG Rickettsiae in ticks collected in 41 counties in Romania. A total of 2028 questing and engorged ticks collected in Romania belonging to five species were tested by PCR amplification of Rickettsia spp. gltA and 17-D gene fragments: Ixodes ricinus (n = 1128), Dermacentor marginatus (n = 507), D. reticulatus (n = 165), Rhipicephalus rossicus (n = 128) and Haemaphysalis punctata (n = 100). Five Rickettsia species were identified following DNA sequence analysis: R. helvetica, R. monacensis, R. slovaca, R. raoultii, and R. hoogstraalii. The most common species detected was R. monacensis. Moreover, R. hoogstraalii was detected for the first time in Romania and in R. rossicus ticks. The detection of R. raoultii and R. monacensis in questing larvae of Hae. punctata suggests the possible transovarial transmission of these Rickettsia species in ticks. The detection of R. hoogstraalii for the first time in Romania increases the reported SFG Rickettsia diversity in the country. [ABSTRACT FROM AUTHOR]

Rhipicephalus bursa is a common tick parasite of small-to-medium size ungulates, principally in warm, temperate, and subtropical areas. Although common in livestock and showing a wide geographic distribution, its epidemiological role in tick-borne bacterial disease is barely known. This study addressed the knowledge gap and aimed to screen for the presence of Anaplasmataceae and spotted fever group (SFG) Rickettsia species in R. bursa ticks collected from domestic animals in Romania, Eastern Europe. A total of 64 pools of R. bursa ticks collected from small ungulates were tested by PCR for Anaplasmataceae DNA presence using group-specific primers. Specific testing was performed for Anaplasma marginale/A. centrale/A. ovis, A. platys, A. phagocytophilum, Ehrlichia canis, and SFG Rickettsia. The positive samples were purified and sequenced, and sequences analysis was used to identify the species and to confirm the PCR results. The only pathogen identified in this study was E. canis. The obtained sequences confirmed the PCR results. The presence of E. canis in R. bursa in Romania and in ticks from sheep was shown for the first time in this study. Based on these findings, it may be presumed that the E. canis DNA originated from ticks; however, the vectorial role of R. bursa (and other arthropod species) in the transmission of E. canis should be proved experimentally. [ABSTRACT FROM AUTHOR]

Despite the increasingly recognized eco-epidemiological importance of ticks as vectors for numerous zoonotic pathogens in urban areas, data regarding the pathogen diversity and co-infection rates in ticks and wildlife hosts in urban and peri-urban Romania are scanty. We aimed to establish the risk of human exposure to co-infected ticks in Cluj-Napoca, a major city in Romania. DNA was isolated from 151 questing ticks: Ixodes ricinus (n = 95), Haemaphysalis punctata (n = 53), Dermacentor reticulatus (n = 2), and Dermacentor marginatus (n = 1); 222 engorged ticks: I. ricinus (n = 164), I. hexagonus (n = 36), H. punctata (n = 16), H. concinna (n = 6), and 70 tissue samples collected from wildlife hosts during 2018 in five urban, and two peri-urban sites. Using a pre-designed Fluidigm real-time PCR dynamic array, all DNA samples were individually screened for the presence of 44 vector-borne pathogens. Subsequently, conventional PCRs were performed for a selection of samples to allow validation and sequencing. In total, 15 pathogens were identified to species and 6 to genus level. In questing ticks, single infections were more common than co-infections. Seven Borrelia spp. were detected in questing I. ricinus , and three in H. punctata ticks. An overall high prevalence 26.35% (95% CI: 19.46–34.22) and diversity of Borrelia burgdorferi sensu lato was seen in urban questing ticks. Other pathogens of the order Rickettsiales were present with variable prevalence. Co-infections occurred in 27.4% (95% CI: 18.72-37.48) of all infected questing ticks. In engorged ticks the overall Bo. burgdorferi sensu lato prevalence was 35.6% (95% CI: 29.29–42.27), with five species present. Pathogens of the order Rickettsiales were also frequently detected. We report for the first time in Romania the presence of Rickettsia aeschlimannii and Rickettsia felis. Overall, from the infected engorged ticks, 69.2% showcased co-infections. In Ixodes spp., dual co-infections, namely Borrelia spp. and Anaplasma phagocytophilum , and Rickettsia helvetica and A. phagocytophilum were the most prevalent. Given the outcome, we underline the need to establish proper tick-surveillance programs in cities and include co-infections in the management plan of tick-borne diseases in Romania. [ABSTRACT FROM AUTHOR]

Anaplasma phagocytophilum and spotted fever group Rickettsia are obligate intracellular Gram-negative tick-borne bacteria, among which several may cause clinical infections in humans. Several Rickettsia spp. and A. phagocytophilum are transmitted in Europe by Ixodes ricinus , the most common tick species feeding on humans in this area. The aim of this study was to evaluate the annual prevalence of Rickettsia spp. and A. phagocytophilum in I. ricinus collected from humans during three consecutive years. The mean prevalences of the infection with the investigated pathogens in I. ricinus ticks collected from human patients were as follows: A. phagocytophilum (5.56%), R. helvetica (4.79%) and R. monacensis (1.53%). In the present study, no significant differences of pathogens prevalence between the three years study period were observed, except the prevalence of R. helvetica , which had a significant increase in 2015, suggesting an increasing risk for humans to be exposed to this zoonotic pathogen. [ABSTRACT FROM AUTHOR]

In Romania, data regarding hard-tick diversity and tick-host associations in wild carnivores are scarce. We aimed to identify tick species in wild carnivores and to establish reliable data on tick-host associations. The study was conducted in various Romanian localities from all five ecoregions found in the country. Fourteen species of wild carnivores were examined. Immature and adult ticks were collected and identified using the morphological keys. The frequency and mean intensity of tick infestation, overall and differentiated by species, developmental stage and host were calculated. Of 202 wild carnivores, 68 were parasitized by seven tick species (predominantly Ixodes ricinus, I. hexagonus and Dermacentor reticulatus). The mean intensity of tick infestation was similar in males (6.97, BCa 95% CI 5.15-9.88) and females (5.76, BCa 95% CI 4.15-9.17). The highest prevalence of infested animals was recorded in the pannonian and steppic ecoregions, 66.7 and 52.7%, respectively. In the continental ecoregion the prevalence was 26.7%, whereas in the pontic ecoregion it was 28%. The lowest value, 16.7% was recorded in the alpine ecoregion. In total 430 ticks were collected, and 24.8% (n = 50) of the animals were infested with more than one tick species. Fourteen new tick-host associations were recorded. Our results suggest that anthropogenic changes of the environment lead to the diminishing of the boundaries, between wild and domestic animals, increasing the exposure for both animals and humans, to infective agents, including tick-borne pathogens. [ABSTRACT FROM AUTHOR]

Background: Anaplasma platys is a bacterium parasitic in the canine platelets, representing the causative agent of canine cyclic thrombocytopenia, with a worldwide distribution, mainly in tropical countries. The agent has zoonotic potential, being reported in several human clinical cases. The suspected vector is the tick Rhipicephalus sanguineus (sensu lato), widely distributed in animals and humans in the tropical regions of South America, Africa, Asia and Australia, but also in southern Europe. Only few scattered data concerning the epidemiology of A. platys in sub-Saharan Africa are available. The aim of our study was to evaluate the epidemiological status of A. platys in dogs and cats from rural communities in eastern and western Africa, where dogs and their ticks live in close contact with humans. Methods: A total of 216 canine and 22 feline blood samples and ticks feeding on them were collected in 2013 and 2014 from eight localities in Ivory Coast and four localities in Kenya. PCR was performed using specific primers targeting a portion of the 16S rRNA gene, followed by sequencing. Results: The main results highlight the high prevalence of A. platys infection in dogs in both Eastern and Western Africa and report it for the first time in Eastern Africa and in Rhipicephalus camicasi. Conclusions: The presence of A. platys DNA in R. camicasi feeding on negative hosts together with the relatively high prevalence of A. platys, correlated with the absence of the probable vector Rhipicephalus sanguineus (s.l.) in Kenyan Island suggest the involvement of other tick species in the transmission of A. platys. [ABSTRACT FROM AUTHOR]

Ticks are medically important vectors of infectious diseases that are able to transmit pathogens to humans and animals. Tick-borne diseases represent a major health concern, posing an increasing risk to the public health during the last century and affecting millions of people. The aim of the current study was to provide epidemiological data regarding the presence of certain tick-borne pathogens in ticks feeding on humans in Romania. Overall, 522 Ixodes ricinus ticks collected from humans were screened for six pathogens: Borrelia spp., Neoehrlichia mikurensis, Babesia spp., Coxiella spp., Bartonella spp., and Francisella tularensis. Ticks attached to humans were collected between 2013–2015 in Cluj County, Romania. Conventional, nested and quantitative PCR were used to detect specific genetic sequences of each pathogen. For identifying the infectious agents, positive samples were sequenced. The infection prevalence was 21.07% from which 8.18% were mixed infections. The detected agents were Borrelia spp., N. mikurensis and Babesia spp. The present data reveal the endemic occurrence of potentially zoonotic pathogens in Romania. Revealing the current distribution of tick-borne pathogens in ticks collected from humans may provide new insights in understanding the complex ecology of tick-borne diseases and enlightens current knowledge about the infection prevalence at local, regional and national levels. [ABSTRACT FROM AUTHOR]