The Presence of Ehrlichia Canis in Rhipicephalus Bursa Ticks Collected from Ungulates in Continental Eastern Europe.

INTRODUCTION: 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. MATERIAL AND METHODS: 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. 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. CONCLUSION: 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.

Human West Nile Meningo-Encephalitis in a Highly Endemic Country: A Complex Epidemiological Analysis on Biotic and Abiotic Risk Factors.

West Nile virus (WNV) is one of the most prevalent mosquito-borne viruses. Although the infection in humans is mostly asymptomatic, 15-20% of cases show flu-like symptoms with fever. In 1% of infections, humans develop severe nervous symptoms and even die, a condition known as West Nile neuroinvasive disease (WNND). The aim of our study was to analyze the influence of abiotic and biotic factors with the human WNND cases during the period 2015-2019. A database containing all the localities in Romania was developed. Abiotic and biotic predictors were included for each locality: geographic variables, climatic data, and biotic factors. Spatial distribution of the WNND infections was analyzed using directional distribution (DD). The Spearman's rank correlation coefficient was employed to assess the strength of association between the WNND infections and predictors. A model was generated using the random forest ensemble learning method. A total number of 535 human WNND cases were confirmed in 308 localities. The DD showed a south-eastern geographical distribution. Weak correlation was observed between the number of human WNND cases for each year and the predictors. The highest predicted probability was around urbanized patches in the south and southeast. Increased surveillance and control measures of vectors in risk areas should be implemented and educational campaigns should be made available for the general public in order to raise awareness of the disease and inform the population about prophylactic measures.

Synopsis of the mosquitoes (Diptera: Culicidae) of Romania.

Mosquitoes (Diptera: Culicidae) are insects capable of transmitting various disease agents. They have a wide geographical range, with a higher diversity and distribution in the tropical and subtropical regions. In Romania, data on mosquito distribution are available mostly from the older literature and published in local journals; hence, it is inaccessible to the international scientific community. The aim of this review was to gather all the published data on mosquitoes in Romania by performing a comprehensive literature search, to create a publicly available georeferenced database for the distribution of mosquitoes and generate distribution maps to identify gaps in the knowledge of mosquito diversity and distribution in the country. The resultant database includes 1,509 unique records for 51 mosquito species representing seven genera: Aedes, Anopheles, Culex, Culiseta, Coquillettidia, Orthopodomyia and Uranotaenia. Knowledge of mosquito distributions is important for understanding the risks of mosquito-borne pathogens present in Romania, a country with high endemicity for various diseases, such as West Nile fever and dirofilariasis. The database also revealed important knowledge gaps, including the lack of data for the Carpathian Arch and the central part of the historical regions of Moldova and Oltenia. The gaps become more evident, as the presence and diversity of the vectors have not been studied in many areas where diseases caused by mosquito-borne pathogens are present. In conclusion, despite the numerous georeferenced records, large areas of Romania remain unexplored for the mosquito species composition, abundance and phenology. This review is a "call for action" for implementing active surveillance in the country.

Environmental factors influencing the distribution of "Theileria annae" in red foxes, Vulpes vulpes in Romania.

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.

Mesocarnivores and macroparasites: altitude and land use predict the ticks occurring on red foxes (Vulpes vulpes).

BACKGROUND: The red fox Vulpes vulpes is the most common mesocarnivore in Europe and with a wide geographical distribution and a high density in most terrestrial habitats of the continent. It is fast urbanising species, which can harbor high numbers of different tick species, depending on the region. Here we present the results of a large-scale study, trying to disentangle the intricate relationship between environmental factors and the species composition of ectoparasites in red foxes. The samples were collected in Transylvania (Romania), a region with a diverse geography and high biodiversity. The dead foxes (collected primarily through the National Surveillance Rabies Program) were examined carefully for the presence of ticks. RESULTS: Ticks (n = 4578) were found on 158 foxes (out of 293 examined; 53.9%). Four species were identified: Dermacentor marginatus, Ixodes canisuga, I. hexagonus and I. ricinus. The most common tick species was I. hexagonus (mean prevalence 37.5%, mean intensity 32.2), followed by I. ricinus (15.0%; 4.86), I. canisuga (4.8%; 7.71) and D. marginatus (3.7%; 3.45). Co-occurrence of two or more tick species on the same host was relatively common (12.6%), the most common co-occurrence being I. hexagonus - I. ricinus. For D. marginatus and I. canisuga the highest prevalence was recorded in lowlands, for I. hexagonus in hilly areas, while for I. ricinus in mountains. CONCLUSIONS: Altitude influenced the intensity of parasitism, with highest intensity observed for all Ixodes species in hilly areas. Dermacentor marginatus occurred only in lowlands, I. canisuga in lowlands and hilly areas while the other two species occurred in all of the regions studied. Foxes from lower altitudes had the most tick species associated, with most incidences of co-parasitism also recorded here. Land use affected tick-species composition, with the presence of D. marginatus strongly associated with the extension of arable areas and lack of forests. The presence of I. hexagonus was determined only by the extent of arable lands. As foxes are frontrunners of wildlife urbanization process, with a continuous increase of their numbers in urban areas, the knowledge of their ticks' ecology (and the pathogens vectored by these) is of utmost importance.

Climate change and species distribution: possible scenarios for thermophilic ticks in Romania.

Several zoonotic tick-borne diseases are emerging in Europe due to various factors, including changes of the cultural landscape, increasing human populations, variation of social habits and climate change. We have modelled the potential range changes for two thermophilic tick species (Hyalomma marginatum and Rhipicephalus annulatus) by use of MaxEnt® and 15 climatic predictors, taking into account the aptitude for future climatic change in Romania. Current models predict increased temperatures, both in the short term (up to 2050) and in the long term (up to 2070), together with possible changes also of the other climatic factors (e.g. precipitation), and may lead to higher zoonotic risks associated with an expansion of the range of the target species. Three different models were constructed (the present, 2050 and 2070) for four representative concentration pathways (RCPs) of greenhouse gas scenarios: RCP2.6, RCP4.5, RCP6, and RCP8.5. The most dramatic scenario (RCP8.5) produced the highest increase in the probable distribution range for both species. In concordance with similar continental-wide studies, both tick species displayed a shift of distribution towards previously cooler areas of Romania. In most scenarios, this would lead to wider ranges; from 9.7 to 43.1% for H. marginatum, and from 53.4 to 205.2% for R annulatus. Although the developed models demonstrate a good predictive power, the issue of species ecology should also be considered.