Fleas of wild carnivores in Romania: diversity, distribution, and host-associations.

BACKGROUND: Fleas are important hematophagous insects, infesting mammals and birds with a worldwide distribution. Fleas of medical importance have been reported from various carnivores worldwide, such as felids, canids, or mustelids. Romania hosts a wide carnivore diversity, but very little is known about flea species that parasitize these animals in Romania. This study aimed to provide a better understanding of the fleas' diversity and their distribution in a relatively large and diverse number of wild carnivore hosts from Romania. METHODS: From 2013 to 2021, 282 carcasses of wild carnivores from different locations in Romania were collected and examined for the presence of ectoparasites. All collected fleas were morphologically identified using specific keys and descriptions. An analysis of the co-occurrence networks was performed. RESULTS: A total of 11 flea species were identified: Pulex irritans (41.09%), Paraceras melis (20.11%), Ctenocephalides felis (7.33%), Ctenocephalides canis (7.83%), Monopsyllus sciurorum (11.11%), Chaetopsylla trichosa (21.96%), Chaetopsylla homoea (5.5%), Chaetopsylla tuberculaticeps (100%), Chaetopsylla rothschildi (13.33%), Chaetopsylla sp. (14.34%), Chaetopsylla globiceps (5.12%), Echidnophaga gallinacea (10%). The statistical analyses showed a significant difference between the infestation of Martes foina with females being more frequently infected than males (66% versus 33%). Paraceras melis infesting Meles meles had a significantly higher prevalence in female badgers than in males (× 2 = 7.7977, P < 0.01) and higher intensities of infestations in males than in females (t = 1.871, P < 0.05). CONCLUSIONS: This is the first large-scale study investigating the distribution and diversity of flea species infesting wild carnivores in Romania. Three flea species were identified for the first time in Romania (E. gallinacea, C. homoea, and C. tuberculaticeps).

Toxoplasma gondii in small mammals in Romania: the influence of host, season and sampling location.

BACKGROUND: Toxoplasma gondii is a protozoan parasite that infects a large spectrum of warm-blooded animals, including humans. Small rodents and insectivores play an important role in the epidemiology of T. gondii and may serve as a source of infection for both, domestic and wild definitive felid hosts. Factors influencing the occurrence of T. gondii in wild small mammals are unknown, despite the fact that many intermediate host species are identified. We have used small mammals (Rodentia and Lipotyphla) captured over two years in various habitats, both in urbanised and in natural landscapes. We assessed the importance of land-use, season and host ecology on T. gondii infection. RESULTS: We examined 471 individuals belonging to 20 small mammal species, collected at 63 locations spread over wide altitude, habitat and land-use ranges from Romania. Heart tissue samples were individually analysed by PCR targeting the 529 bp repetitive DNA fragment of T. gondii. The overall prevalence of infection was 7.3%, with nine species of rodents and two species of shrews being found to carry T. gondii DNA. Five species showed high frequency of infection, with the highest prevalence found in Myodes glareolus (35.5%), followed by Spermophilus citellus (33.3%), Sorex minutus (23.1%), S. araneus (21.7%) and Micromys minutus (11.1%). Adults seemed more often infected than young, however when controlling for season, the difference was not significant, as in spring both adults and young showed higher infection rates, but more adults were sampled. Contrary to our expectations, urban/rural areas (with their implicit high density of domestic feline presence) had no effect on infection prevalence. In addition, neither habitat, nor land-use at sampling sites was important as only geographical location and host species were contributing factors to the infection risk. CONCLUSIONS: High prevalence of T. gondii infection showed a highly localised, patchy occurrence, with long living and higher mobility host species being the most common carriers, especially during autumn.

The Role of Wild Boars in the Circulation of Tick-Borne Pathogens: The First Evidence of Rickettsia monacensis Presence.

Most wild mammals can serve as hosts both for tick-borne pathogens (TBPs) and for the ticks themselves. Among these, wild boars, due to their large body size, habitat and life span, show high exposure to ticks and TBPs. These species are now one of the widest-ranging mammals in the world, as well as the most widespread suid. Despite the fact that certain local populations have been decimated by African swine fever (ASF), wild boars are still considered overabundant in most parts of the world, including Europe. Altogether, their long-life expectancy, large home ranges including migration, feeding and social behaviors, wide distribution, overabundance and increased chances of interactions with livestock or humans make them suitable sentinel species for general health threats, such as antimicrobial-resistant microorganisms, pollution and ASF geographical distribution, as well as for the distribution and abundance of hard ticks and also for certain TBPs, such as Anaplasma phagocytophilum. The aim of this study was to evaluate the presence of rickettsial agents in wild boars from two counties in Romania. Among 203 blood samples of wild boars (Sus scrofa ssp. attila) collected during 3 (2019-2022) hunting seasons (September-February), 15 were found positive for tick-borne pathogen DNA. Six wild boars were positive for A. phagocytophilum DNA presence and nine for Rickettsia spp. The identified rickettsial species were R. monacensis (six) and R. helvetica (three). No animal was positive either for Borrelia spp., Ehrlichia spp. or Babesia spp. To the best of our knowledge, this is the first report of R. monacensis in European wild boars, thus adding the third species from the SFG Rickettsia, in the epidemiology of which this wild species may have a role as a reservoir host.

The Effect of Extraction Methods on Phytochemicals and Biological Activities of Green Coffee Beans Extracts.

The objectives of the present study consisted of identifying the impact of extraction methods and parameters held over the phytochemistry and biological activities of green coffee beans. Extraction processes belonging to two categories were performed: classical methods-maceration, Soxhlet extraction, and such innovative methods as turboextraction, ultrasound-assisted extraction, and a combination of the latter two. Total polyphenolic and flavonoid content, as well as in vitro antioxidant activity of the resulted extracts were spectrophotometrically determined. Extracts displaying the highest yields of bioactive compounds were subjected to High Performance Liquid Chromatography-Mass Spectrometry analysis. The extracts with the best phytochemical profiles were selected for biological activity assessment. In vivo, a model of plantar inflammation in Wistar rats was used to determine antioxidant activity, by evaluating the oxidative stress reduction potential, and anti-inflammatory activity. In vitro antimicrobial activity was also determined. The Soxhlet extraction and ultrasound-assisted extraction gave the highest bioactive compound yields. The highest total polyphenolic content was 2.691 mg/mL gallic acid equivalents and total flavonoid content was 0.487 mM quercetin equivalents for the Soxhlet extract subjected to 60 min extraction time. Regarding the antioxidant activity, ultrasound-assisted extraction reached the highest levels, i.e., 9.160 mg/mL Trolox equivalents in the DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) assay and 26.676 mM Trolox equivalents in the FRAP (Ferric Reducing Antioxidant Power) assay, at a 30 min extraction time and 50 °C extraction temperature. The 60 min Soxhlet extract reached the highest level for the ABTS+ (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) assay, 16.136 mM Trolox equivalents, respectively. Chlorogenic acid was present in the highest concentration in the same Soxhlet extract, 1657.179 µg/mL extract, respectively. Sterolic compounds were found in high concentrations throughout all the analyzed extracts. A proportional increase between yields and extraction parameter values was observed. Increased inhibition of Gram-negative bacteria was observed. The finally selected Soxhlet extract, that of 60 min extraction time, presented a significant in vivo antioxidant activity, with a slight anti-inflammatory activity. Antioxidant levels were elevated after 2 h of extract administration. Pro-inflammatory cytokine secretion was not influenced by the administration of the extract.

Milk Pathogens in Correlation with Inflammatory, Oxidative and Nitrosative Stress Markers in Goat Subclinical Mastitis.

Goat mastitis is still frequently diagnosed in dairy farms, with serious consequences on milk quality and composition. The aim of this study was to establish correlations between milk microorganisms and biochemical parameters in goats with no signs of clinical mastitis. Thus, 76 milk samples were collected from a dairy goat farm, Carpathian breed, followed by microbiological, molecular (16S rRNA sequencing) and somatic cells analysis, determination of lactate dehydrogenase (LDH), ß-glucuronidase, catalase (CAT), glutathione peroxidase (GPx) activity, total antioxidant capacity (TAC), nitric oxide (NO) and lipid peroxides (LPO) using spectrophotometry and the ELISA method for 8-hydroxy-deoxyguanosine (8-OHdG) as the oxidative DNA damage indicator. Samples positive for bacterial growth showed a significant (p < 0.05) increase in the number of somatic cells, LDH and ß-glucuronidase activity, as well as higher levels of CAT, GPx, NO, LPO and 8-OHdG compared with pathogen-free milk whereas TAC was lower in milk from an infected udder. These findings suggest that subclinical mastitis is associated with increased enzymatic activity and induction of oxidative stress. Nevertheless, changes in biochemical parameters tended to vary depending on the pathogen, the most notable mean values being observed overall in milk positive for Staphylococcus aureus.

Spotted Fever Group Rickettsia spp. Diversity in Ticks and the First Report of Rickettsia hoogstraalii in Romania.

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.

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.

Anaplasma phagocytophilum in Multiple Tissue Samples of Wild Carnivores in Romania.

Wild vertebrate hosts can serve as reservoirs or amplification hosts for tick-borne pathogens (TBPs). Wild carnivores due to their large size have an increased risk for harboring large numbers of ticks. Moreover, their large home ranges and long lives may increase the risk of exposure to ticks and TBPs. Wild carnivores therefore may be good sentinel species with which to monitor the distribution of TBPs. We aimed to evaluate the presence of rickettsial DNA in wild carnivores and to compare its presence in different types of samples. In total, 95 wild carnivores from nine species, originating from 17 counties of Romania collected during 2014-18, were included in the study. From each animal, DNA was extracted from multiple tissue samples, including blood clot, heart, liver, lungs, spleen, kidney, lymph node, and bone marrow, and screened for the presence of rickettsial pathogen DNA (Anaplasma phagocytophilum, Ehrlichia canis, and Rickettsia spp.). Samples from 10 animals from six species (Canis aureus, Ursus arctos, Canis lupus, Felis sylvestris, Lutra lutra, and Martes foina) were found to be positive for A. phagocytophilum. The most frequently positive sample was the spleen. No animal was positive for Ehrlichia spp. or Rickettsia spp. Wild carnivores may be involved in the ecoepidemiology of A. phagocytophilum by maintaining the infection in synanthropic environments.

Biologically Active Extracts from Different Medicinal Plants Tested as Potential Additives against Bee Pathogens.

Honey bees (Apis mellifera) perform pollination service for many agricultural crops and contribute to the global economy in agriculture and bee products. However, honey bee health is an ongoing concern, as illustrated by persistent local population decline, caused by some severe bee diseases (e.g., nosemosis, AFB, EFB, chalkbrood). Three natural recipes are in development based on the bioactive compounds of different plants extract (Agastache foeniculum, Artemisia absinthium, Evernia prunastri, Humulus lupulus, Laurus nobilis, Origanum vulgare and Vaccinium myrtillus), characterised by HPLC-PDA. The antimicrobial activity of these recipes was tested in vitro against Paenibacillus larvae, Paenibacillus alvei, Brevibacillus laterosporus, Enterococcus faecalis, Ascosphaera apis and in vivo against Nosema ceranae. A mix of 20% blueberry, 40% absinthium, 10% oakmoss, 10% oregano, 10% Brewers Gold hops, 5% bay laurel and 5% anise hyssop extract showed the strongest antibacterial and antifungal activity. Combing several highly active plant extracts might be an alternative treatment against bee-disease-associated parasites and pathogens, in particular to replace synthetic antibiotics.

Prevalence of Antimicrobial Resistant Bacteria from Conjunctival Flora in an Eye Infection Prone Breed (Saint Bernard).

The conjunctival bacterial resident and opportunistic flora of dogs may represent a major source of dissemination of pathogens throughout the environment or to other animals and humans. Nevertheless, contamination with bacteria from external sources is common. In this context, the study of the antimicrobial resistance (AMR) pattern may represent an indicator of multidrug resistant (MDR) strains exchange. The present study was focused on a single predisposed breed-Saint Bernard. The evaluated animals were healthy, but about half had a history of ocular disease/treatment. The swabs collected from conjunctival sacs were evaluated by conventional microbiological cultivation and antimicrobial susceptibility testing (AST). The most prevalent Gram-positive was Staphylococcus spp.; regardless of the history, while Gram-negative was Pseudomonas spp.; exclusively from dogs with a history of ocular disease/treatment. Other identified genera were represented by Bacillus, Streptococcus, Trueperella, Aeromonas and Neisseria. The obtained results suggest a possible association between the presence of mixed flora and a history of ocular disease/treatment. A high AMR was generally observed (90%) in all isolates, especially for kanamycin, doxycycline, chloramphenicol and penicillin. MDR was recorded in Staphylococcus spp. and Pseudomonas spp. This result together with a well-known zoonotic potential may suggest an exchange of these strains within animal human populations and the environment.

Efficacy of Natural Formulations in Bovine Mastitis Pathology: Alternative Solution to Antibiotic Treatment.

INTRODUCTION: Bovine mastitis is an inflammatory disease of the udder that causes important economic losses in the animal breeding and dairy product industries. Nowadays, the conventional livestock antibiotic treatments are slowly being replaced by alternative treatments. In this context, the main aim of this study was to evaluate the efficacy of natural products in alternative treatment of bovine mastitis. MATERIAL AND METHODS: Two natural formulations with previously suggested in vitro antimicrobial effect were tested in vivo on mastitic cows. Animals with a positive diagnosis for mastitis (n = 20) were divided into three treatment groups: two groups (n = 8) were administered formulations of propolis, alcoholic extracts of Brewers Gold and Perle hops, plum lichen, common mallow, marigold, absinthe wormwood, black poplar buds, lemon balm, and essential oils of oregano, lavender, and rosemary designated R4 and R7 (differing only in the latter being more concentrated) and one group (n = 4) a conventional antibiotic mixture. In vivo efficacy of treatments was evaluated by somatic cell and standard plate counts, the treatment being considered efficacious when both parameters were under the maximum limit. RESULTS: R7 was effective in the most cases, being therapeutically bactericidal in six out of eight cows, while R4 gave good results in three out of eight cows, and conventional antibiotics cured one out of four. CONCLUSION: These results suggest the possible therapeutic potential of these natural products in bovine mastitis.

Detection of Leishmania infantum DNA and antibodies against Anaplasma spp., Borrelia burgdorferi s.l. and Ehrlichia canis in a dog kennel in South-Central Romania.

Canine vector-borne diseases are caused by pathogens transmitted by arthropods including ticks, mosquitoes and sand flies. Many canine vector-borne diseases are of zoonotic importance. This study aimed to assess the prevalence of vector-borne infections caused by Dirofilaria immitis, Ehrlichia canis, Borrelia burgdorferi sensu lato, Anaplasma spp. and Leishmania infantum in a dog kennel in Argeș County, Romania. Dog kennels are shelters for stray dogs with no officially registered owners that are gathered to be neutered and/or boarded for national/international adoptions by various public or private organizations. The international dog adoptions might represent a risk in the transmission of pathogens into new regions. In this context, a total number of 149 blood samples and 149 conjunctival swabs from asymptomatic kennel dogs were assessed using serology and quantitative real-time polymerase chain reaction. Antibodies against B. burgdorferi s.l. were detected in one dog (0.6%), anti-Anaplasma antibodies were found in five dogs (3.3%), while ten dogs (6.7%) tested positive for D. immitis antigen. Overall, 20.1% (30/149) of dogs were positive for L. infantum DNA. All samples were seronegative for anti-Leishmania antibodies. When adopting dogs from this region of Romania, owners should be aware of possible infection with especially L. infantum. The travel of infected dogs may introduce the infection to areas where leishmaniasis is not present.

Multiple Tick-Borne Pathogens in Ixodes ricinus Ticks Collected from Humans in Romania.

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.

Design, Synthesis and Biological Evaluation of New Piperazin-4-yl-(acetyl-thiazolidine-2,4-dione) Norfloxacin Analogues as Antimicrobial Agents.

 In an effort to improve the antimicrobial activity of norfloxacin, a series of hybrid norfloxacin-thiazolidinedione molecules were synthesized and screened for their direct antimicrobial activity and their anti-biofilm properties. The new hybrids were intended to have a new binding mode to DNA gyrase, that will allow for a more potent antibacterial effect, and for activity against current quinolone-resistant bacterial strains. Moreover, the thiazolidinedione moiety aimed to include additional anti-pathogenicity by preventing biofilm formation. The resulting compounds showed promising direct activity against Gram-negative strains, and anti-biofilm activity against Gram-positive strains. Docking studies and ADMET were also used in order to explain the biological properties and revealed some potential advantages over the parent molecule norfloxacin.

Borrelia spp. in small mammals in Romania.

BACKGROUND: Small mammals play an important role in the life-cycle of ticks and are reservoirs for several zoonotic pathogens. The aim of this study was to provide epidemiological data regarding the presence of Borrelia spp. in tissues of small mammals from Romania. METHODS: We examined 401 individuals belonging to 11 small mammal species collected in Romania. Collections cover the largest effort to survey these reservoirs in the country. Tissue samples were analyzed by multiplex qPCR targeting the ospA gene of Borrelia burgdorferi (s.l.) and a part of the flaB gene of B. miyamotoi. Positive samples were further analysed by conventional PCR and sequenced. RESULTS: The overall prevalence of infection with Borrelia spp. in small mammal tissues was 4.9%. The most commonly detected species were B. afzelii, followed by B. garinii/B. bavariensis, B. miyamotoi and B. burgdorferi (s.s.). To our knowledge, we report for the first time the detection of Borrelia spp. in Crocidura leucodon and C. suaveolens, and B. miyamotoi in the liver of Myodes glareolus. CONCLUSIONS: To our knowledge, our study evaluates for the first time the occurrence of Borrelia spp. in small mammals in Romania, contributing to a better knowledge of the distribution of these bacteria. This survey upgrades previous data on the spatial distribution of the pathogens and reveals the importance of animal surveillance regarding Lyme borreliosis and relapsing fever caused by B. miyamotoi.

Thelazia callipaeda, an Endemic Parasite of Red Foxes ( Vulpes vulpes) in Western Romania.

Thelazia callipaeda (Spirurida, Thelaziidae) is a vector-borne zoonotic nematode parasite of the conjunctival sac of a large spectrum of hosts, currently having an expanding distribution range throughout Europe. In Romania, the parasite has a relatively large distribution range and host spectrum. However, the presence of T. callipaeda in red foxes ( Vulpes vulpes) has not yet been confirmed. The aim of the present study was to assess the prevalence and geographical distribution of T. callipaeda in red foxes from Romania. A total of 514 animals originating from 10 counties of western Romania were evaluated for the presence of T. callipaeda. Overall, 151 red foxes (29.38%) were positive. No significant differences were noticed between sex and age groups of positive foxes. In total, 3,350 adult nematodes were recovered. The intensity of infection had a mean value of 23.2, while the sex ratio was 2.2:1 in favor of females. In 91 foxes, both eyes were affected, while 60 harbored unilateral infection. Sequence analysis of the cox 1 gene of one nematode from each positive fox ( n=151) revealed the exclusive presence of haplotype h1. We report a relatively high prevalence and distribution range of T. callipaeda infection in red foxes in Romania, highlighting their potential role as a wildlife reservoir.

New records for Anaplasma phagocytophilum infection in small mammal species.

BACKGROUND: Tick-borne diseases pose a major threat in public health. The epidemiological dynamics of these diseases depends on the tick vector species and their hosts, as well as the geographical distribution and ecology of both. Among many possible hosts for ticks, small mammals have a major role in the development of immature stages of several tick species. Small mammals are also important reservoir hosts for several pathogenic agents and possible reservoirs for Anaplasma phagocytophilum. In this context, the aim of our study was to evaluate the prevalence of A. phagocytophilum in small mammal species in Romania. RESULTS: A total of 791 small mammals of 31 species were tested by PCR, targeting the rrs gene for detection of A. phagocytophilum DNA. Positive results were obtained in 20 small mammals: five Apodemus flavicollis (6.49%), three Sorex araneus (9.09%), three A. uralensis (4.84%), two A. sylvaticus (3.92%), and one of each Spermophilus cittelus (7.14%), Microtus agrestis (3.85%), Sorex minutus (3.85%), Muscardinus avellanarius (3.13%), Crocidura suaveolens (2.44%), Mus spicilegus (2%) and M. arvalis (1.75%). CONCLUSIONS: Eleven small mammal species were found to be carriers of A. phagocytophilum, suggesting a possible involvement of these species in its epidemiology. To our knowledge, this is the first report of A. phagocytophilum in S. minutus, C. suaveolens, M. spicilegus, M. avellanarius and S. citellus.

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.

Babesia vesperuginis, a neglected piroplasmid: new host and geographical records, and phylogenetic relations.

BACKGROUND: Babesia spp. are hemoparasites which infect the red blood cells of a large variety of mammals. In bats, the only known species of the genus is Babesia vesperuginis. However, except a few old reports, the host range and geographical distribution of this bat parasite have been poorly studied. This study aimed to investigate the presence of piroplasms in tissues of bats collected in four different countries from eastern and central Europe: Austria, Czech Republic, Hungary and Romania. METHODS: A total of 461 bat carcasses (24 species) were collected between 2001 and 2016 from caves, mines and buildings. PCR was performed using specific primers targeting a portion of the 18S rDNA nuclear gene and cytochrome c oxidase subunit 1 mitochondrial gene, followed by sequencing. RESULTS: The results of this study show for the first time the presence of B. vesperuginis in bats in central and eastern Europe. The phylogenetic analysis of the 18S rDNA nuclear gene revealed no variability between the sequences and the phylogenetic analysis of the cox1 mitochondrial gene proved that B. vesperuginis could be divided into two subclades. CONCLUSION: Our study showed a broad geographical distribution of B. vesperuginis in European bats, reporting its presence in five new host species (M. cf. alcathoe, M. bechsteinii, M. myotis, Pi. nathusii and V. murinus) and three new countries.

Filarioid infections in wild carnivores: a multispecies survey in Romania.

BACKGROUND: Filarioids are vector-borne parasitic nematodes of vertebrates. In Europe, eight species of filarioids, including zoonotic species, have been reported mainly in domestic dogs, and occasionally in wild carnivores. In Romania, infections with Dirofilaria spp. and Acanthocheilonema reconditum are endemic in domestic dogs. Despite the abundant populations of wild carnivores in the country, their role in the epidemiology of filarioid parasites remains largely unknown. The aim of the present study was to assess the host range, prevalence and distribution of filarioid infections in wild carnivores present in Romania. METHODS: Between May 2014 and February 2016, 432 spleen samples originating from 14 species of wild carnivores have been tested for the presence of DNA of three species of filarioids (D. immitis, D. repens and A. reconditum). RESULTS: Overall 14 samples (3.24%) were molecularly positive. The most prevalent species was D. immitis (1.62%), accounting for 50% (n = 7) of the positive animals. The prevalence of D. repens was 1.39%, while that of A. reconditum was 0.23%. No co-infections were detected. Dirofilaria immitis DNA was detected in five golden jackals, Canis aureus (7.58%), one red fox, Vulpes vulpes (0.33%), and one wildcat, Felis silvestris (10%). The presence of D. repens DNA was detected in two red foxes (0.66%), two golden jackals (3.03%), one grey wolf (7.14%), and one least weasel, Mustela nivalis (33.33%). Acanthocheilonema reconditum DNA was found only in one red fox (0.33%). CONCLUSION: The present study provides molecular evidence of filarial infections in wild carnivore species in Romania, suggesting their potential epidemiological role and reports a new host species for D. repens.