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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.

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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.

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&ndash, 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.

The objective of this study was to determine the occurrence of Staphylococcus spp., Escherichia coli, somatic coliphages, F-specific RNA bacteriophages, hepatitis E virus (HEV), and bovine enterovirus (BEV) in fecal and water samples. The occurrence of Staphylococcus spp. was investigated in treated wastewater samples collected from slaughterhouse of Tunisia. Results showed that Staphylococcus spp. were detected in the totality of collected samples with an average mean of 5.44 Log10 (CFU/100 ml). Regarding fecal indicator bacteria, E. coli was detected in the totality of water samples and was more abundant in Tunisian samples than in samples collected from Romania (P

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.

Canine granulocytic anaplasmosis (CGA) is an important tick-borne disease with worldwide distribution. The importance of this disease resides in the ability of Anaplasma phagocytophilum to infect humans and several animal species. The aim of the study was to evaluate the prevalence rate of CGA in different altitudinal areas of Romania. A total of 357 canine blood samples were collected during 2010-2013 from eight counties. To assess the influence of the altitude on A. phagocytophilum prevalence, the samples were collected from four different altitude areas (coastal 0-5 meters; lowland 6-100 meters; hilly areas 200-300 meters; low mountain areas >500 meters). These samples were evaluated for the presence of A. phagocytophilum DNA by amplifying part of the Ankyrin repeat protein (AnkA) gene. A higher prevalence was obtained for coastal compared with remaining areas, suggesting an influence of altitude on the CGA. Moreover, the results suggest an influence of climate and rainfall. In the present research work, we highlight the risk of granulocytic anaplasmosis in Central and Southern Romania, with a greater risk associated to Southern lowland region, especially in coastal areas. The importance of these results resides in the zoonotic potential of the canine A. phagocytophilum strains. In conclusion, the altitude and precipitation level may be risk factors for A. phagocytophilum infection in dogs and other hosts, including humans.

An extensive survey of parasites in red foxes ( Vulpes vulpes ) was independently conducted in Romania and the Czech Republic. Carcasses were examined by necropsy, and small, dark nodules apparently containing ticks were noticed in the subcutaneous tissue of several foxes. Histopathological examination was performed using hematoxylin and eosin (HE) staining. Of the 91 foxes examined from the Czech Republic, 14 (15.4%) were harboring ticks in the subcutaneous tissue. In the majority of these cases, 1–3 nodules/fox were found, with a maximum of 31 nodules/fox. In Romania a single examined fox had subcutaneous ticks. All ticks collected from subcutaneous tissue were partially engorged adults. Based on morphological features, Ixodes ricinus , I. hexagonus , I. crenulatus and Dermacentor reticulatus were identified. The histopathological examination revealed chronic granulomatous panniculitis with peripheral fibrosis and intralesional presence of the ticks. Only few data are available regarding ticks localized in the subcutaneous tissue of any host. All the ticks were dead or already decomposed and it is evident that subcutaneous location does not represent an evolutionary advantage, as the detachment and finishing the life cycle is impossible.

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.

The genus Anaplasma (Rickettsiales: Anaplasmataceae) comprises obligate intracellular Gram-negative bacteria that are mainly transmitted by ticks, and currently includes six species: Anaplasma bovis, Anaplasma centrale, Anaplasma marginale, Anaplasma phagocytophilum, Anaplasma platys, and Anaplasma ovis. These have long been known as etiological agents of veterinary diseases that affect domestic and wild animals worldwide. A zoonotic role has been recognized for A. phagocytophilum, but other species can also be pathogenic for humans. Anaplasma infections are usually challenging to diagnose, clinically presenting with nonspecific symptoms that vary greatly depending on the agent involved, the affected host, and other factors such as immune status and coinfections. The substantial economic impact associated with livestock infection and the growing number of human cases along with the risk of transfusion-transmitted infections, determines the need for accurate laboratory tests. Because hosts are usually seronegative in the initial phase of infection and serological cross-reactions with several Anaplasma species are observed after seroconversion, direct tests are the best approach for both case definition and epidemiological studies. Blood samples are routinely used for Anaplasma spp. screening, but in persistently infected animals with intermittent or low-level bacteremia, other tissues might be useful. These guidelines have been developed as a direct outcome of the COST action TD1303 EURNEGVEC (>European Network of Neglected Vectors and Vector-Borne Diseases>). They review the direct laboratory tests (microscopy, nucleic acid-based detection and in vitro isolation) currently used for Anaplasma detection in ticks and vertebrates and their application., This work was done under the frame of COST action TD1303.

Anaplasma phagocytophilum is the agent of tick-borne fever, equine, canine and human granulocytic anaplasmosis. The common route of A. phagocytophilum transmission is through a tick bite, the main vector in Europe being Ixodes ricinus. Despite the apparently ubiquitous presence of the pathogen A. phagocytophilum in ticks and various wild and domestic animals from Europe, up to date published clinical cases of human granulocytic anaplasmosis (HGA) remain rare compared to the worldwide status. It is unclear if this reflects the epidemiological dynamics of the human infection in Europe or if the disease is underdiagnosed or underreported. Epidemiologic studies in Europe have suggested an increased occupational risk of infection for forestry workers, hunters, veterinarians, and farmers with a tick-bite history and living in endemic areas. Although the overall genetic diversity of A. phagocytophilum in Europe is higher than in the USA, the strains responsible for the human infections are related on both continents. However, the study of the genetic variability and assessment of the difference of pathogenicity and infectivity between strains to various hosts has been insufficiently explored to date. Most of the European HGA cases presented as a mild infection, common clinical signs being pyrexia, headache, myalgia and arthralgia. The diagnosis of HGA in the USA was recommended to be based on clinical signs and the patient’s history and later confirmed using specialized laboratory tests. However, in Europe since the majority of cases are presenting as mild infection, laboratory tests may be performed before the treatment in order to avoid antibiotic overuse. The drug of choice for HGA is doxycycline and because of potential for serious complication the treatment should be instituted on clinical suspicion alone.

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.

Bovine mastitis is a cause of major financial losses in the dairy industry worldwide. Bovine mastitis is defined as the inflammatory response of the udder tissue due to either physical trauma or infections caused by different microorganisms, especially bacteria. More than 135 pathogenic agents have been identified from bovine mastitis. The aim of the study was to identify the bacterial genera involved in mastitis etiology in cattle and to test the antimicrobial susceptibility to antibiotics. The samples were collected before morning milking and evaluated by microscopic and cultural examination, and further identified using biochemical methods. The evaluation of antimicrobial susceptibility was performed using Kirby-Bauer disk diffusion test. Overall, Staphylococcus spp., Bacillus spp., Streptococcus spp., Micrococcus spp., Escherichia spp. and Trueperella spp. were identified. The most frequent bacterial associations were observed for Staphylococcus-Bacillus, Bacillus-Streptococcus and Staphylococcus-Micrococcus. Regarding the antimicrobial sensitivity profile, the majority of the bacterial strains we have isolated were classified as sensitive, with the exception of Escherichia coli, with one strain being resistant to all tested antibiotics. Among the antimicrobials, the Amoxicillin and clavulanic acid was the most efficient one and Trimethoprim/Sulfamethoxazole did not exhibit an inhibitory activity in 19 milk samples.

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.

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. Electronic supplementary material The online version of this article (10.1186/s13071-017-2536-3) contains supplementary material, which is available to authorized users.

Bovine mastitis a major disease that is commonly associated with bacterial infection. The common treatment is with antibiotics administered intramammary into infected quarters of the udder. The excessive use of antibiotics leads to multidrug resistance and associated risks for human health. In this context, the search for alternative drugs based on plants has become a priority in livestock medicine. These products have a low manufacturing cost and no reports of antimicrobial resistance to these have been documented. In this context, the main objective of this study was to determine the antimicrobial effect of extracts and products of several indigenous, or acclimatized plants on pathogens isolated from bovine mastitis. A total of eleven plant alcoholic extracts and eight plant-derived products were tested against 32 microorganisms isolated from milk. The obtained results have shown an inhibition of bacterial growth for all tested plants, with better results for Evernia prunastri, Artemisia absinthium, and Lavandula angustifolia. Moreover, E. prunastri, Populus nigra, and L. angustifolia presented small averages of minimum inhibitory and bactericidal concentrations. Among the plant-derived products, three out of eight have shown a strong anti-microbial effect comparable with the effect of florfenicol and enrofloxacin, and better than individual plant extracts possibly due to synergism. These results suggest an important anti-microbial effect of these products on pathogens isolated from bovine mastitis with a possible applicability in this disease.

Background Dirofilaria immitis and Dirofilaria repens are mosquito-borne zoonotic filarioids typically infecting dogs, causing a potentially fatal cardiopulmonary disease and dermatological conditions, respectively. The females are larviparous, releasing the larvae (microfilariae) into the bloodstream, which further develop in mosquito vectors. However, microfilaremia greatly fluctuates during a 24-h period. As the sampling time can greatly influence the accuracy of diagnosis, the aim of the present study was to assess the circadian periodicity of D. immitis and D. repens in naturally co-infected dogs in an endemic area of Romania and to investigate possible differences of periodicity between these two species. Methods Overall, four dogs harbouring natural co-infection with D. immitis and D. repens were selected and sampled every two hours for two consecutive days: two dogs in July 2014 and two in July 2015. At each sampling time, a 0.7 ml blood sample was taken. Modified Knott’s test was performed on 0.5 ml, and the remaining 0.2 ml were used for DNA extraction and molecular amplification, both in single and duplex PCR reactions. Microfilariae of both species were morphologically identified and counted in each collected sample, microfilaremia was calculated, and fluctuation was charted. Results The dynamics of microfilaremia showed similar patterns for both Dirofilaria species. In all four dogs, D. immitis was present at all sampling times, with several peak values of microfilaremia, of which one was common for all dogs (1 am), while minimum counts occurred between 5 and 9 am. Similarly, for D. repens, one of the peak values was recorded in all dogs at 1 am, while minimum counts (including zero) occurred at 9 and 11 am. Single species-specific PCR reactions were positive for both D. immitis and D. repens in all collected samples, while duplex PCR failed to amplify D. repens DNA in many cases. Conclusions Both Dirofilaria immitis and D. repens microfilariae are subperiodic, following a similar variation pattern, with peak values of microfilaremia registered during the night in Romania. Duplex PCR fails to identify the infection with D. repens in co-infected dogs when the ratio of microfilaremia is in favour of D. immitis. Electronic supplementary material The online version of this article (doi:10.1186/s13071-017-2055-2) contains supplementary material, which is available to authorized users.

&nbsp, In an effort to improve the antimicrobial activity of norfloxacin, a series of hybrid norfloxacin&ndash, 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.

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.

Anaplasma platys, the aetiological agent of infectious canine cyclic thrombocytopaenia, infects platelets of dogs, usually causing mild or asymptomatic disease. Although A. platys is transmitted by ticks, as for other Anaplasma species, alternative modes of transmission may be involved. The aim of the present study was to evaluate the occurrence of A. platys infection in litters of puppies, which could suggest possible vertical transmission. Twelve litters, together with the respective bitches, were included in the study for the detection of A. platys DNA by PCR, followed by sequencing. Five puppies, from 2/4 litters

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. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1443-3) contains supplementary material, which is available to authorized users.

During the last decades, Dirofilaria spp. infection in European dogs has rapidly spread from historically endemic areas towards eastern and northeastern countries, but little or no information is available from these geographical regions. The present study provides a picture of filarial infections in dogs from Romania and compares two tests for the diagnosis of Dirofilaria immitis. From July 2010 to March 2011, blood samples were collected from 390 dogs from nine counties of Romania and serological SNAP tests were performed for the detection of D. immitis antigen. The remaining blood clots were subsequently used for DNA extraction followed by multiplex PCR for assessing filarioid species diversity (i.e. D. immitis, Dirofilaria repens and Acanthocheilonema reconditum). Based on molecular detection, an overall prevalence of 6.92 % (n = 27; 95 % confidence interval (CI) 4.70-10.03 %) for D. repens, 6.15 % (n = 24; 95 % CI 4.07-9.14 %) for D. immitis and 2.05 % (n = 8; 95 % CI 0.96-4.16 %) for A. reconditum was recorded, with significant variations according to sampling areas. Coinfections of D. immitis and D. repens were recorded in 23.91 % (n = 11) positive dogs. A slightly higher prevalence for D. immitis was detected at the SNAP test (n = 28, 7.17 %; 95 % CI 4.91-10.33 %), but this difference was not statistically significant (p = 0.66). However, only 53.57 % (n = 15) of antigen-positive dogs were confirmed by PCR, while other dogs (n = 9) PCR positive for D. immitis were negative at the serology. The present study shows that Dirofilaria species are endemic in the southern and southeastern areas of Romania, This article also provides, for the first time, an epidemiological picture of the distribution of A. reconditum in Romania.

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.

Among wildlife, the red foxes (Vulpes vulpes) are one of the most adapted wild species to anthropic ecosystems. They are well recognized as important reservoirs for a large number of zoonotic agents in Europe, including ticks and tick-borne pathogens. Currently, there are few available data on the importance of red foxes in the ecoepidemiology of vector-borne diseases. Therefore, the aim of this study was to screen the dynamics of tick infestation in 357 red foxes from 12 Romanian counties. Tick identification was performed using the morphological keys. The overall prevalence of tick infestation was 43.7%. The 5753 collected ticks belonged to five species: Ixodes hexagonus (on 113 out of 156 foxes; prevalence 72.44%), I. ricinus (28.84%), I. crenulatus (7.7%), Dermacentor marginatus (7.05%) and Haemaphysalis punctata (0.64%). Coinfestation occurred in 24 foxes (22 with 2 tick species; 2 with 3 tick species) with the following associations: I. ricinus + I. hexagonus (n = 10), I. hexagonus + D. marginatus (n = 5), I. ricinus + I. crenulatus (n = 4), I. ricinus + D. marginatus (n = 2), I. hexagonus + I. crenulatus (n = 1), D. marginatus + I. hexagonus + I. ricinus (n = 1), and H. punctata + I. hexagonus + I. ricinus (n = 1). This study indicates that foxes are hosts to a relevant number of tick species with recognized vectorial role. The provided information can facilitate the understanding of the ecology of ticks and can be the basis for studies on the epidemiology of tick-borne diseases. The high prevalence of tick infestation in red foxes, coupled with the increasing presence of this species in synanthropic environments and a more nature-oriented lifestyle of people, can pose a significant threat to human health, through the emergence of tick-borne diseases. Moreover, foxes represent a good model of sentinel species. This research was performed as part of project IDEI PCE 236/2011.

Background Angiostrongylus daskalovi is a rare cardiopulmonary nematode infecting badgers. The parasite was described in 1988 and, since then, found only once in mustelids in Europe. The present study aims to report new cases of patent A. daskalovi infection in badgers from northern Romania and to provide new information on its ultrastructure, molecular diagnosis, and pathology. Methods Eight road-killed or hunted badgers originating from Maramureș and Alba counties in Romania were collected and necropsied. Adults and larvae of cardio-pulmonary nematodes were collected and examined by light and scanning electron microscopy (SEM). Genomic DNA was extracted from adults and first-stage larvae (L1). PCR amplification of the internal transcribed spacer 2 (ITS2, ∼500 bp) of the rRNA gene was performed. Amplicons were purified, sequenced, and compared to those available in the GenBank database. Histopathological examination of the lungs was performed and lesions described. Results The necropsy revealed the presence of nematodes in the pulmonary arteries of three animals. All parasites were mature adults and the coproscopic examination showed the presence of eggs and L1 larvae in all three positive animals. Light microscopy examination confirmed the morphological and morphometric similarity of parasites to Angiostrongylus daskalovi. SEM highlighted the typical angiostrongylid structure of the rays of the copulatory bursa and the anterior extremity, with the presence of six sensory papillae surrounding the mouth opening in which a triangular tooth was visible. The first-stage larva (L1) of A. daskalovi is described here for the first time. Histopathological examination of the lungs showed chronic interstitial verminous pneumonia due to the presence of adult parasites. Molecular analysis showed 100 % nucleotide similarity to an Angiostrongylus sp. isolate originating from a badger from Spain, tentatively identified as A. daskalovi. Conclusions Our study unequivocally demonstrates the presence of A. daskalovi in European badgers from Romania, provides the first description of the larvae and reveals new data about the ultrastructure of adult parasites and their pathological impact, contributing to the understanding of the phylogenetic relationships with other congeneric species.

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 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.