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Background Ticks are hematophagous arthropods which normally attach to the surface of the host’s skin. Their aberrant presence in the subcutaneous tissue of a few carnivores, predominantly foxes, has been reported. However, there have been no reports of this phenomenon in other carnivores such as mustelids or golden jackals. Our aim was to investigate the host spectrum for this aberrant localization of ticks. Methods Between 2015 and 2020, a total of 198 carcasses of 12 species of carnivore were examined by parasitological necropsy. When a subcutaneous tick was found, the nodule was removed, carefully dissected, and stored in ethanol. The morphological identification of the subcutaneous tick was carried out to species level. Results A single subcutaneous tick was found in one carcass, that of a golden jackal (Canis aureus). The tick was identified as a female Ixodes ricinus. All the other carcasses were negative for the presence of subcutaneous ticks. Conclusion To our knowledge, this is the first report of a subcutaneous tick in a golden jackal. This finding broadens the host spectrum of subcutaneous ticks, and reinforces the idea that, among carnivores, this phenomenon only occurs in canids. Graphical Abstract

Haemoparasites of the genus Babesia infect a wide range of domestic and wild animals. Feline babesiosis is considered endemic in South Africa, while data on Babesia spp. infection in felids in Europe is scarce. Using samples from 51 wild felids, 44 Felis silvestris and 7 Lynx lynx, the study aimed to determine the presence and genetic diversity of Babesia spp. in wild felids in Romania by analyzing the 18S rDNA and two mitochondrial markers, cytochrome b (Cytb) and cytochrome c oxidase subunit I (COI) genes. By 18S rDNA analyses, Babesia spp. DNA was detected in 20 European wild felids. All sequences showed 100% similarity to B. canis by BLAST analysis. Conversely, Cytb and COI analyses revealed the presence of two Babesia spp., B. pisicii n. sp., which we herein describe, and B. canis. The pairwise comparison of both mitochondrial genes of B. pisicii n. sp. showed a genetic distance of at least 10.3% from the most closely related species, B. rossi. Phylogenetic analyses of Cytb and COI genes revealed that B. pisicii n. sp. is related to the so-called “large” canid-associated Babesia species forming a separate subclade in a sister position to B. rossi.

In March 2017, a captive male juvenile (ca. 6 months old) olive baboon (Papio anubis) was brought to a primate rescue center in Benin with multiple subcutaneous swellings of unknown aetiology. At the general inspection of the body, around 15 partially mobile masses of variable sizes were found in different locations across the body. Following two surgical procedures, several cyst-like structures were removed and placed either in 10% formalin or in absolute ethanol. The cysts had a typical coenurus-like morphology. Genomic DNA was extracted from one cyst using a commercially available kit. The molecular characterization was performed by PCR amplification and sequencing of a region of the nuclear ITS-2 rDNA and a fragment of the mitochondrial 12S rDNA gene, revealing its identity as T. serialis, with 88%–98% similarity to T. serialis isolates from USA, Japan and Germany This study reports a new host for the larval form of T. serialis and confirms the role of primates as intermediate host, highlighting also the risk for human infection. Further genetic studies from multiple geographic isolates are needed to clarify the taxonomic status of this group., Graphical abstract Image 1, Highlights • The present study reports a the olive baboon as new host for the larval form of Taenia serialis. • This is the first report of T. serialis in Benin. • The relevance and risk for the health and welfare of primates in rescues centers is discussed.

Dirofilaria immitis is a worldwide spread nematode affecting the pulmonary artery and the heart of dogs (rarely reported in cats), especially in areas where the dogs show a high prevalence of infection. Angiostrongylus chabaudi is, in turn, a cardiopulmonary nematode of felids identified in several southern European countries and Germany. Co-infections of Dirofilaria immitis and Angiostrongylus spp. are known only in canids. We report a case of D. immitis and A. chabaudi co-infection in a road-killed wildcat originating from Southeastern Romania. Overall, 17 nematodes were collected from the pulmonary arteries of the wildcat and were morphologically identified as A. chabaudi (2 males and 15 females). Another nematode was collected from the right ventricle and identified as adult male D. immitis. Genomic DNA was extracted from one nematode of each of the two species and a fragment of the cox1 gene was amplified and sequenced. The sequences obtained from the nematodes showed 100% similarity to a sequence of A. chabaudi isolated from Romania (Accession number KU521521) and to various D. immitis sequences from Europe, Asia and Australia (e.g. KT716014, EU159111, AJ537512). This is the first report of A. chabaudi and D. immitis co-infection in a felid, revealing the possibility of similar situations in domestic cats. This requires a more in-depth clinical and laboratory examination of animals with respiratory and cardiac symptoms.

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.

Bovine thelaziosis can be caused by Thelazia rhodesi Desmarest 1828, Thelazia gulosa Railliet & Henry 1910 and Thelazia skrjabini Erschow 1928 which are known to be transmitted by species of Muscidae. Therapeutic measures in cattle include mechanical removal of the adult parasites, followed by irrigation of the conjunctival sac with different solutions such as levamisole (1%) or administration of ivermectin (0.2 mg/kg SC), doramectin (0.2 mg/kg SC or pour-on) both with high efficacy, however, the use of such macrocyclic lactones can be problematic in lactating cattle. Nematodes harboured in the eyes of some cattle from Romania were morphologically identified as Thelazia rhodesi and the PCR analysis confirmed the diagnosis with a 98.0% nucleotide similarity for ITS1 sequence, with other sequences available in GenBank. The present paper reports the presence of T. rhodesi in cattle from a dairy farm in Romania and the recovery after treatment with eprinomectin. To the best of our knowledge this is the first report on the efficacy of eprinomectin against Thelazia spp. and the first molecular confirmation of T. rhodesi 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&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.

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.

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.

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.

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.

The risk of developing Lyme borreliosis (LB) after the bite of a Borrelia (B.) burgdorferi sensu lato (s.l.) infected tick in Romania is unknown.The present prospective study, performed in 2010-2011 in a hospital in Romania, has followed-up clinical and serological outcome of patients that presented with B. burgdorferi positive Ixodes (I.) ricinus bite. A second group of patients, including age, sex and residence-matched individuals bitten by B. burgdorferi negative ticks, was followed-up as a control group. The subjects' outcome was evaluated one year after the tick bite.Forty-three out of 389 ticks detached from patients were positive by hbb Real-Time PCR (RT-PCR) for B. burgdorferi s.l. (mainly B. afzelii, but also B. garinii, B. burgdorferi sensu stricto, B. spielmanii/B. valaisiana and B. lusitaniae). Twenty patients bitten by B. burgdorferi positive ticks and twenty matched control patients returned for the one year follow-up. Two patients from the B. burgdorferi positive group developed clinical manifestations of acute LB (erythema migrans) and 5 patients seroconverted (two from the B. burgdorferi positive group and three from the B. burgdorferi negative group). Borrelia afzelii was identified in ticks collected from persons that developed erythema migrans (EM). Comparing the two groups of patients, no statistical significant differences were found regarding presence of clinical symptoms or seroconversion.No outcome differences were found between the group of patients bitten by B. burgdorferi positive ticks and the group of patients bitten by B. burgdorferi negative ticks.

The objective of this study was to evaluate different methods used for detection of Borrelia burgdorferi sensu lato (s.l.) in ticks: immunohistochemistry followed by focus floating microscopy (FFM) and real-time polymerase chain reaction (real-time PCR) targeting the ospA and hbb genes. Additionally, an optimized ospA real-time PCR assay was developed with an integrated internal amplification control (IAC) for the detection of inhibition in the PCR assay and was validated as an improved screening tool for B. burgdorferi. One hundred and thirty-six ticks collected from humans in a hospital from Cluj-Napoca, Romania, were investigated regarding genus, stage of development and sex, and then tested by all three assays. A poor quality of agreement was found between FFM and each of the two real-time PCR assays, as assessed by concordance analysis (Cohen's kappa), whereas the agreement between the two real-time PCR assays was moderate. The present study argues for a low sensitivity of FFM and underlines that discordant results of different assays used for detection of B. burgdorferi in ticks are frequent.

Cysticercosis is a zoonotic disease that may have a significant impact on the public health. The aim of the current study was to assess the seroprevalence of porcine cysticercosis in Northwestern Romania and the influence of several risk factors such as age, sex and season. The study included 1,288 pigs originating from 7 different counties and was conducted between May 2014 and May 2015. All pigs were reared in the traditional backyard pig system. Of these, 226 were young and 1,062 were adult pigs, 188 were males and 1,100 females. Serum samples were collected. The apDia Cysticercosis Antigen ELISA (apDia, Belgium) commercial kit was used to identify the viable cysticerci of Taenia sp. Out of the 1,288 serum samples, 83 were seropositive (seroprevalence: 6.4%). The risk factors’ analysis showed that age (3.1% juveniles vs 7.1% adults [P < 0.03]) and season (9.3% fall vs 11.8% winter [P P P = 0.1). Considering the genus specificity of ELISA commercial kit, the serology should be correlated with the post mortem examination in order to confirm the disease and have a Cysticercus cellulosae specific prevalence.

Background There is a need for experimental animal models for inflammatory bowel diseases (IBD), but no proposed model has been unanimously accepted. Objectives The aim of this study was to develop 2 affordable models of IBD in rats and to compare them. Material and methods We produced IBD in rats using either dextran sodium sulfate (DSS) or 2, 4, 6-trinitrobenzene sulfonic acid (TNBS). The requirements for experimental models were: a predictable clinical course, histopathology and inflammation similar to human ulcerative colitis (UC) and Crohn's disease (CD). The effect of acute administration of DSS and TNBS on oxidative stress (as measured by the assessment of glutathione peroxidase - GPx) was verified. The activity of whole blood GPx was measured using a commercially available Randox kit (Crumlin, UK). Results The administration of DSS increased GPx activity compared to the control and TNBS-treated groups, but not to a statistically significant degree. Histological examination of the colonic mucosa following the administration of DSS showed multifocal erosions with minimal to mild inflammatory infiltrate, mainly by polymorphonuclear cells (PMN), lymphocytes and plasma cells. For TNBS-induced colitis, the histological changes manifested as multifocal areas of ulcerative colitis with mild to severe inflammatory infiltrate. Whole blood GPx values displayed a direct dependence on the chemical agent used. Our results show a correlation between histopathology, proinflammatory state and oxidative stress. Conclusions The experimental DSSor TNBS-induced bowel inflammation used in this study corresponds to human IBD and is reproducible with characteristics indicative of acute inflammation in the case of the protocols mentioned.

Background Giardia duodenalis is a parasitic protist that infects a large number of species, being localized in the small intestine. Two of the eight recognized assemblages have zoonotic potential, but studies regarding their distribution in less important pet or farm species are scarce. Of these species, the long-tailed chinchilla is a host for Giardia spp., although data on the spread of infection and assemblages involved are confined. The present work aimed to determine the prevalence of Giardia infection and assemblage identification in farmed chinchillas in Romania. A total of 341 fecal samples were collected from 5 farms and microscopically examined using flotation test based on saturated sodium chloride solution. DNA from all positive samples was extracted and identified by PCR targeting the gdh gene. Results The overall prevalence of Giardia infection was 55.7% (190/341); there was no statistically significant difference (P = 0.25) in prevalence between young animals (58.8%) and adults (52.6%). Assemblages B (151/190), D (33/190) and E (6/190) were identified. Among assemblage B, sub-assemblages BIII (6/151) and BIV (145/151) were determined. Conclusions This study demonstrates that Giardia spp. infection is highly prevalent in farmed chinchillas from Romania, and the sub-assemblages identified are potentially zoonotic.

In this study, Hepatozoon species was molecularly identified and characterized for the first time on the Indian Ocean island of Mauritius. Partial sequences of the 18S rRNA gene of the Hepatozoon isolates were analysed from three naturally infected dogs. The sequences of H. canis were similar to the 18S rRNA partial sequences (JX112783, AB365071 99%) from dog blood samples from West Indies and Nigeria. Our sequences were deposited in the GenBank database.

Trichinella are nematodes parasitic in the skeletal muscles of terrestrial vertebrates, generally transmitted via predatorism. It is expected that the infection would have certain influences on the muscular activity in infected animals. The aim of the study was to develop an experimental model for studying the muscular effort in laboratory mice prior to the experimental infection with Trichinella and to evaluate the method in trained (with free access to a voluntary activity wheel) and untrained (without access to activity wheel) animals. Ten laboratory mice (all adult males), equally divided in two groups were used: a control group (untrained mice) and a second group (trained). The weight was evaluated individually. The muscular activity was evaluated using an effort-wheel. Values were expressed in instantaneous power (IP) and data were recorded using a constant speed of 5 rpm for 5 and 20 minutes. The instantaneous power (IP) developed by the effort wheel at 5 minutes was significantly lower in the control group than in trained mice. Similar results were obtained for the maximum power (MP). Interestingly, for the trained mice, there was no difference between the average IP at 5 and 20 minutes of activity. The results show the utility of trained mice, establish the necessary experiment time and validate the method for evaluating the influence of Trichinella spp. on the muscular activity of experimentally infected mice.

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.

To the Editor: Ixodes spp. ticks are vectors for human and animal pathogens. Ix. ricinus ticks are widely distributed, frequently reported to feed on humans, and the main vector for a large variety of tickborne pathogens (1). The effect of ticks and tickborne diseases on public health, animal health and welfare, and animal production appears to be an increasing global problem, which will lead to considerable economic costs (2).

The aim of the present experiment was to assess the clinical, haematological, biochemical and immunological responses of pigs experimentally infected with Trichinella spiralis. One group of 6 pigs was infected with 1500 larvae/kg body weight and one group of 7, was maintained as control. The highest larval density was in the diaphragm (504.3 ± 8.2). During the experimental T. spiralis infection in pigs, increased values of total granulocyte number, eosinophils, blood glucose, K+ concentration, CK, ALT, AST and ALP were registered in different stages of infection. A slight increase of the phagocytic activity was registered 14 days p.i., in the infected group. Significantly increased values in lymphocyte activity, in infected group, was observed 30 and 60 days p.i. comparative with control. The specific IgG antibodies were detectable by ELISA at 14 days p.i. The results obtained in this study provide a better understanding about complete interactions occurring during Trichinella infection in pigs.

Ticks and tick-borne diseases represent a great concern worldwide. Despite this, in Romania the studies regarding this subject has just started, and the interest of medical personnel, researchers, and citizens is increasing. Because the geographical range of many tick-borne diseases started to extend as consequences of different biological and environmental factors, it is important to study the diversity of ticks species, especially correlated with host associations. A total number of 840 ticks were collected between 1 April and 1 November 2010, from 66 animals, from 17 species in 11 counties, spread all over Romania. Four Ixodidae species were identified: Dermacentor marginatus (49.2%), Ixodes ricinus (48.3%), Hyalomma marginatum (2.4%), and Rhipicephalus sanguineus (0.1%). The obtained results indicate that D. marginatus is the most abundant tick species and I. ricinus is the most prevalent. As both of them are important vectors for human and animal diseases, the present paper discusses the associated risks for tick-borne diseases.

Previous studies in southeastern Europe reported a high incidence of human trichinellosis and a high prevalence of Trichinella infection in animals in countries like Bulgaria, Croatia, Romania and Serbia. The aim of this study was, using routine Trichinella test data in pig and game animals, to investigate the extent of Trichinella infection in slaughtered animals in Romania, over the period of 1997–2004, and to identify possible differences in prevalence among the various regions of Romania. Trichinella infection data were obtained from trichinelloscopic examinations of domestic (backyard and industrial reared pigs) and game species (wild boar and bears). A marked difference between Transylvania and other counties of Romania, observed for human trichinellosis, was taken into account when analyzing Trichinella epidemiological data. A cumulative prevalence of 8 cases/10 4 animals tested for pigs, 9 cases/10 3 animals tested for wild boars, and 13.1 cases/10 2 animals tested for bears was obtained for the 8 years period. Analysis of animal prevalence data demonstrated a geographical heterogeneity: whereas Trichinella prevalence in pigs is much lower in Transylvania than in the other counties, Trichinella prevalence in game animals is similar for the different regions. This observation suggests that, in Romania, rather than the levels of the parasite circulating in domestic and game animals, it was changes in the social and political structure of Romania in the 1990s, combined with inadequate meat inspection practices that were the main contributors to these geographic variations.

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.

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.

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

Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum are two tick-borne pathogens of medical and/or veterinary importance which are distributed worldwide. Erinaceus roumanicus , the Northern white-breasted hedgehog, is a common synanthropic species that is known to carry not only the hedgehog tick, Ixodes hexagonus , but also I. ricinus , the most common European tick species. I. ricinus is the main vector of both mentioned pathogens. Within this framework and because only limited information is available on the role of E. roumanicus in the ecology of B. burgdorferi s.l. and Anaplasma phagocytophilum in Europe, we carried out an epidemiological surveillance on this species in Romania. From the 57 examined hedgehogs collected in 12 different counties, 24 presented tick infestation. Most ticks ( n = 959) were morphologically identified as larvae, nymphs, or adults of I. ricinus . The prevalence of B. burgdorferi s.l. was 0.4%, and that of A. phagocytophilum 12%. In all positive cases for B. burgdorferi s.l., restriction fragment length polymorphism revealed the genospecies B. afzelii . In Romania, only limited information is available on the epidemiology of B. burgdorferi s.l. and A. phagocytophilum . As hedgehogs commonly share the same environment with humans and other potential reservoir hosts for tick-borne pathogens, our study provides new epidemiological data of public health importance.

Herbivorous animals are usually, by virtue of their diet, outside the major transmission cycles of Trichinella spp. However, since 1975, the year of the first report of human trichinellosis caused by the consumption of infected horse meat, the domestic horse has appeared as a novel vector of Trichinella spp. infection to humans, with 15 outbreaks documented in France and Italy. Romania, one of the main countries exporting horses into the European Union (EU), experienced a dramatic increase of Trichinella spp. infection in both domestic pigs and humans in the 1990s. Some Trichinella spiralis-infected horses were exported to the EU during this period. The aim of this study was to evaluate the prevalence of Trichinella spp. infections in horses from Romania using both direct and indirect tests. Of 3000 serum samples tested in 2001, none were positive by ELISA using three different Trichinella antigens (crude; excretory/secretory, ES; stg-BSA antigens). Of 2992 serum samples tested in 2002, 17 (0.56%) showed optical density values higher than the cut-off in an ELISA using ES antigens and one was confirmed by western blot (WB). Four of the 17 ELISA positive horses, including the horse with a confirmed serology by WB, were subjected for intensive meat examination at slaughter, but no Trichinella spp. larvae were detected. Further, no Trichinella spp. larvae were detected by trichinelloscopy and artificial digestion of 25,838 horses slaughtered in Alexandria and Timisoara between 2001 and 2004. The false positive results obtained by serology confirm the previous work on the unreliability of serology for detection of Trichinella spp. infection in horses. Furthermore, the lack of detection of Trichinella spp. infected horses by artificial digestion, suggests a very low prevalence of infection in horses in Romania.

In order to evaluate prevalence and intensity of apicomplexan hemoparasites in free-ranging reptiles from Romania, blood smears were collected from European pond turtles (Emys orbicularis), sand lizards (Lacerta agilis), and spur-thighed tortoises (Testudo graeca). All three host species were positive for blood parasites, with prevalence of infected individuals between 60.71% and 100% and variable intensity. Similarities and differences with other epidemiological data are discussed.

International audience; According to the International Commission on Trichinellosis survey in 2004, Romania has the most cases of trichinellosis in the world. Epidemiologic data for each county were collected and analyzed from two different time periods: before (1980–1989) and after (1990–2004) political changes. Data were analyzed separately for Transylvania and the rest of the Romanian counties. During the past 25 years, 28,293 human cases of trichinellosis were reported with an incidence of 51.0 cases per 106 persons per year. An important increase in the incidence was observed from 1980 to 1989 compared with the 1990–2004 period. For the entire period, the incidence rate obtained for Transylvanian counties (82.2 cases per 106 persons per year) was higher than the incidence rate obtained for the other counties (35.7 cases per 106 persons per year). Hypotheses and facts contributing to the heterogeneity of human trichinellosis cases are discussed.

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.