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Parasitic diseases cause significant economic losses in swine, including free-range swine farms, the number of which in Romania has increased in the last decades. The current study aimed to identify the parasitic profile of swine raised on two free-range (low-input) farms from Transylvania. Nine hundred sixty samples collected from weaners, fatteners, and sows were investigated by flotation, centrifugal sedimentation, modified Ziehl-Neelsen stained fecal smear, modified Blagg technique, and oocyst/egg cultures. The number of oocysts (OPG), cysts (CPG), and eggs (EPG) were counted per gram of fecal matter. The examination revealed parasitic infections with Balantidium coli, Eimeria spp., Ascaris suum, Trichuris suis, Oesophagostomum spp., Strongyloides ransomi and Cryptosporidium spp. Prevalence (P) and the mean intensity (MI) of the infections varied according to age, swine category, farm, and season. The overall prevalence in both free-range farms according to the age category was 63.2%—Eimeria spp., 70.31%—B. coli, 9.38%—Oesophagostomum spp., 3.75% S. ransomi, and 18.12% Cryptosporidium spp. in weaners. In fatteners Eimeria spp. revealed a prevalence of 50.93%, B. coli—72.5 %, A. suum—63.13%, T. suis—39.06%, and in sows Eimeria spp.—39.06%, B. coli—62.19%, A. suum—34.06%, Oesophagostomum spp.—27.19%, S. ransomi—1.56% and Cryptosporidium spp.—9.38%. The study revealed statistically significant (p < 0.05) differences between age groups, seasons, and farms for all diagnosed parasites. Further research is required to better understand the epidemiology of these infections in swine from Transylvania.

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

To understand the importance of host’ habitat choice in determining parasite burden, we studied the distribution of two helminth parasites of the red fox (Vulpes vulpes) in south-eastern Europe (Romania): Crenosoma vulpis and Eucoleus aerophilus, both widely distributed respiratory nematodes parasitic in various carnivores. Even though the life cycle and biology of the two nematodes follow a different pattern, both parasites appear to be co-distributed and often co-infect foxes with variable prevalences across their range. Between July 2016 and August 2018, 550 red foxes, V. vulpes were collected by hunters in different localities from 22 counties of Romania and examined by necropsy. All parasites found in the trachea and bronchial system were collected and preserved in 70% ethanol. We characterised red fox/parasite habitats using seven predictors (fragmentation, altitude, presence/absence of water surface, per cent cover of arable land/grassland/urbanized areas/forest cover/wetlands). Prevalence, abundance, intensity, and sex ratio were calculated and statistically analysed using the R software. Out of the 550 examined foxes, 76.2% were infected with lungworms. The overall prevalence was 32.0% for C. vulpis and 72.5% for E. aerophilus. The mean intensity of infection was 13.70 for C. vulpis 6.15 for E. aerophilus. For both nematodes, the prevalence was significantly higher in males than in females, and there was no influence of hosts’ age. No statistical differences were found for intensity and mean intensity in the case of infection with C. vulpis and E. aerophilus between age and sex categories. The abundance of C. vulpis showed a strong positive relationship with the presence of wetlands and habitat fragmentation. We found a significant correlation between the abundance of E. aerophilus and altitude, with foxes from higher elevations showing higher prevalences., Graphical abstract Image 1, Highlights • Crenosoma vulpis and Eucoleus aerophilus are widely distributed respiratory nematodes. • Prevalence, abundance, intensity were calculated and statistically analysed using R software Grs. • There was a significant correlation between the abundance of E. aerophilus and altitude. • The abundance of C. vulpis showed a strong positive relationship with the presence of wetland.

Nematodes of the genus Trichinella are among the most widespread parasites of domestic and wild omnivores and predatory animals. The present study aimed to evaluate the antiparasitic effect of Lactobacillus casei ATCC 393 (original) and L. paracasei CNCM in CD-1 mice experimentally infected with Trichinella britovi. Four groups of 20 mice (10 females and 10 males/group) were used, with two control (C) groups and two experimental (E) groups, in which each animal received a daily oral dose of 100 µL of 105 CFU/mL probiotics in Ringer’s solution. On day 7, all mice (except the negative control group) were infected orally with Trichinella (100 larvae/animal) as well as the two probiotics. On day 9 post-infection (p.i.), 10 mice/group were euthanized, and the presence of adult parasites in the intestinal content and wall was tested. On day 32 p.i., 10 mice/group were euthanized, then trichinoscopy and artificial digestion were performed to assess the muscle infection with T. britovi. On day 9 p.i., the experimental group pretreated with L. casei ATCC 393 (6.3 ± 3.03) showed a significantly lower number of adult parasites in the intestinal wall compared with the positive control group (24.6 ± 4.78). Additionally, a significantly lower adult parasite count in the intestinal wall was registered in female mice pretreated with L. paracasei CNCM (7.4 ± 4.71) compared to female mice from the positive control (29.0 ± 5.17). No statistically relevant results were obtained concerning the male mice or the data obtained at 32 days p.i., irrespective of mice gender.

Background The population of wild felids is large and stable in Romania with many carnivore habitats being protected. Felids can be infected with a wide variety of cardio-pulmonary nematodes and can act as reservoirs of infection for domestic cats. The aim of this study was to evaluate the distribution and species diversity of cardio-pulmonary nematodes in wild felids from Romania. Methods A total of 54 wild felids (7 Lynx lynx and 47 Felis silvestris) were legally collected from different locations in Romania and analysed by complete parasitological necropsy. The entire respiratory tract was longitudinally opened and checked for the presence of nematodes. Detected nematodes were collected and morphologically identified to species level. Results Two Eurasian lynxes and 29 European wildcats were positive for cardio-pulmonary nematodes. Eurasian lynxes were infected with two species of cardio-pulmonary nematodes, Eucoleus aerophilus and Troglostrongylus brevior, while in wildcats the dominant parasite was E. aerophilus (34.0%) followed by Angiostrongylus chabaudi (23.4%) and T. brevior (14.9%). Dirofilaria immitis and Aelurostrongylus abstrusus were each detected in two wildcats (4.3%). Conclusions The present study expanded the epidemiological knowledge on felid cardiopulmonary nematodes in Romania. We confirmed the presence of A. abstrusus in wildcats and a patent infection with T. brevior in Eurasian lynx. Graphical Abstract

Thelaziosis caused by Thelazia callipaeda is an emerging disease in Europe. Only two reports of naturally infected lagomorphs have been published so far. The aim of this study was to evaluate the status of the Romanian populations of European brown hares, Lepus europaeus as reservoir hosts for T. callipaeda. Between November 2019 and November 2021, the eyes of 326 L. europaeus carcasses were examined for the presence of ocular parasites. Nematodes were stored in plastic vials with physiological saline, followed by morphological and molecular identification. QGis 3.20 and EpiInfoTM 7 were used for mapping and statistical analysis. Four (1.23%) hares harbored T. callipaeda infection, with a total of 84 nematodes collected (mean intensity 21 nematodes/host), with 45 males, 39 females (two sexually immature, seven with only eggs, and 30 with eggs and larvae). One specimen from each host was successfully sequenced resulting in a 100% similarity with several other sequences of T. callipaeda haplotype 1. Statistical analysis revealed no significant results. The current study represents a first report of T. callipaeda in the European brown hare in Romania, and the second in Europe, also reiterating the role of lagomorphs as reservoir hosts for this zoonotic ocular nematode.

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.

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.

Dirofilaria immitis is an important mosquito-borne nematode, being of both veterinary and public health concern. The typical final host is represented by the domestic dog, Canis familiaris, but it is able to infect a large variety of mammalian species. During the present study (March 2016–February 2022), a total of 459 wild carnivore carcasses belonging to 17 species, from Romania, were evaluated for the presence of adult D. immitis by necropsy. Overall, 20 animals (4.36%) were positive: twelve golden jackals, C. aureus (19.05%); four red foxes, Vulpes vulpes (6.67%); one raccoon dog, Nyctereutes procyonoides; two wild cats, Felis silvestris (4.65%); and one European badger, Meles meles (0.87%). This study provides further evidence of the occurrence of the canine heartworm, D. immitis, in Romania, expanding the known host spectrum, reports a new host species for this parasite, the European badger, and a new host for Europe, the raccoon dog.

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. Electronic supplementary material The online version of this article (10.1186/s13071-018-2791-y) contains supplementary material, which is available to authorized users.

Background The genus Troglostrongylus includes nematodes infecting domestic and wild felids. Troglostrongylus brevior was described six decades ago in Palestine and subsequently reported in some European countries (Italy, Spain, Greece, Bulgaria, and Bosnia and Herzegovina). As the diagnosis by the first-stage larvae (L1) may be challenging, there is a possibility of confusion with Aelurostrongylus abstrusus. Hence, the knowledge on the distribution of this neglected feline parasite is still scarce. The present paper reports the first case of T. brevior infection in Romania. In July 2017, a road-killed juvenile male Felis silvestris, was found in in Covasna County, Romania. A full necropsy was performed and the nematodes were collected from the trachea and bronchioles. Parasites were sexed and identified to species level, based on morphometrical features. A classical Baermann method was performed on the lungs and the faeces to collect the metastrongyloid larvae. Genomic DNA was extracted from an adult female nematode. Molecular identification was accomplished with a PCR assay targeting the ITS2 of the rRNA gene. Results Two males and one female nematodes were found in the trachea and bronchioles. They were morphologically and molecularly identified as T. brevior. The first-stage larvae (L1) recovered from the lung tissue and faeces were morphologically consistent with those of T. brevior. No other pulmonary nematodes were identified and no gross pulmonary lesions were observed. Conclusions This paper represents the first report of Troglostrongylus brevior infection in Romania, so far representing the second northernmost location for this genus in Europe. The diversity of species infecting wild and domestic felids and the differences regarding the clinical significance of these nematodes highlight the need for a more intense surveillance and proper diagnosis of feline lungworm infections, especially in countries where more species were demonstrated to be present. Furthermore, an increased awareness between clinicians is needed for a correct diagnostic approach to feline lungworm diagnosis.

SUMMARYNematodes of the Angiostrongylidae family, such as Angiostrongylus vasorum and Angiostrongylus cantonensis, may cause potentially life-threatening diseases in several mammal species. Alongside these well-known species, Angiostrongylus chabaudi has been recently found affecting the cardiopulmonary system of domestic and wild cats from Italy, Germany, Greece, Romania and Bulgaria. Nonetheless, significant gaps in the understanding of A. chabaudi epidemiology include the lack of information of species acting as intermediate host and of the morphological description of larval stages. Cornu aspersum (n = 30) land snails were infected with 100 first-stage larvae of A. chabaudi collected from a naturally infected wildcat in Romania. Larvae at different developmental stages were found in 29 out of 30 (96·7%) infected snails and a total of 282 (mean 9·8 ± 3·02 larvae per each specimen) were collected from the gastropods. Here we demonstrate that A. chabaudi develops in snails and report C. aspersum as potential intermediate host for this parasitic nematode. Findings of this study are central to understand the ecological features of feline angiostrongylosis and its epidemiology within paratenic and intermediate hosts.

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.

Background Jackals are medium-sized canids from the wolf-like clade, exhibiting a unique combination of ancestral morphotypes, broad trophic niches, and close phylogenetic relationships with the wolf and dog. Thus, they represent a potential host of several pathogens with diverse transmission routes. Recently, populations of the Eurasian golden jackal Canis aureus have expanded into the Western Palaearctic, including most of Europe. The aim of our study was to examine Eurasian golden jackals from Romania, Czech Republic and Austria for a wide spectrum of vector-borne protists and to evaluate the role of this species as a reservoir of disease for domestic dogs and/or humans. Results Diagnostic polymerase chain reaction (PCR) DNA amplifications revealed 70% of jackals to be positive for Hepatozoon, 12.5% positive for piroplasms, and one individual positive for Leishmania infantum. Phylogenetic analyses of partial 18S rDNA sequences invariably placed sequenced isolates of Hepatozoon into the H. canis clade. For piroplasms, both the 18S and cox1 sequences obtained confirmed the presence of Babesia canis and “Theileria annae” in 5 and 2 individuals, respectively, providing the first records of these two piroplasmids in Eurasian golden jackals. A single animal from Dolj County (Romania) was PCR-positive for L. infantum, as confirmed also by sequencing of ITS1-5.8S. Conclusions Apparently, expanding populations of jackals can play a significant role in spreading and maintaining new Babesia canis foci in Central Europe. The role of jackals in the epidemiology of “Theileria annae” and H. canis is probably similar to that of red foxes and should be taken into account in further research on these parasites. Also the presence of L. infantum deserves attention. Our study confirms that once established, the populations of Eurasian golden jackals constitute natural reservoirs for many canine vector-borne diseases, analogous to the role of the coyotes in North America.

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.

Red foxes (Vulpes vulpes (L.)) are widespread across Europe, tolerant of synanthropic ecosystems, and susceptible to diseases potentially shared with humans and other animals. We describe flea fauna on red foxes in Romania, a large, ecologically diverse country, in part because fleas may serve as an indicator of the risk of spillover of vector-borne disease. We found 912 individual fleas of seven species on the 305 foxes assessed, for an infestation prevalence of 49.5%. Mean flea load per fox was 5.8 (range 0-44 fleas), and flea detections were most abundant in fall and early spring. Fleas included generalists (Ctenocephalides canis (Curtis), 32.6% of all fleas), Ct. felis (Bouché, 0.1%), and Pulex irritans L. (29.9%), the fox specialist Chaetopsylla globiceps (Taschenberg, 32.5%), mesocarnivore fleas Paraceras melis Walker (3.2%) and Ch. trichosa Kohaut (1.5%), and the small mammal flea Ctenophthalmus assimilis (Taschenberg, 0.1%), which is rarely or never reported from carnivores. There were significantly more female than male Ch. globiceps, Ct. canis, and Pu. irritans, and these three species were the most broadly distributed geographically. Diversity indices suggested reduced diversity in mountainous areas above 700 m. When compared to other flea studies on foxes in Europe, Romania had flea diversity near the median of reports, which was unexpected given Romania's high ecological diversity. Notably absent prey specialists, compared to other studies, include Archaeopsylla erinacei (Bouché) and Spilopsyllus cuniculi (Dale). Further studies of possible disease agents in fox fleas could help elucidate possible risks of vector-borne disease in foxes, domestic animals, and humans as well.

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.

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.

Nematodes of genus Trichinella are wide-spread zoonotic parasites, able to infect a large variety of vertebrates. Animal hosts are usually regarded as asymptomatic carriers. However, there is little data regarding the functional consequences that T. spiralis infection renders on muscle cells. The aim of the present study was to assess the effect of T. spiralis on the effort capacity of experimentally infected mice. Overall, 60 mice, divided into three groups were used: M (uninfected), L200 and L1000, infected with 200 or 1000 larvae/mouse respectively. The mice were periodically weighed and their effort capacity was evaluated (days 0, 7, 15, 35 and 60). From each group, two randomly selected mice were euthanized after evaluation carcasses were artificially digested in order to establish the number of larvae per gram (LPG). On day 0, there were no significant differences among groups. Starting with day 7, the effort capacity of infected groups decreased, with significant differences between group M and the infected groups. From day 15, the differences between the infected groups also became significant. The LPG gradually increased and the differences between groups were always significant. A strong correlation between the LPG and decreased effort capacity was noted. The present study demonstrates the reduction of muscular capacity in mice experimentally infected with Trichinella spiralis, in correlation with the infective dose, providing new insights in this parasite's transmission strategy.

Background Thelazia callipaeda (Spirurida, Thelaziidae) is a vector-borne zoonotic eye worm with a broad host spectrum. In Europe, it is an emerging threat, having greatly expanded its geographical distribution during the past two decades. In Romania, T. callipaeda has been previously reported in domestic and wild canids and felids. The aim of the present study was to assess the occurrence of T. callipaeda in mustelids in the country. Methods Between March 2015 and April 2019, 77 road-killed mustelids (3 pine martens, Martes martes; 6 European polecats, Mustela putorius; 13 beech martens, Martes foina; and 55 European badgers, Meles meles) were examined by necropsy. If present, all ocular nematodes were collected and stored in absolute ethanol, for subsequent morphological and molecular identification. Results Two animals were found to be infected with T. callipaeda: one European badger and one beech marten. The molecular analysis revealed a 100% nucleotide similarity to T. callipaeda haplotype h1 for all the sequenced specimens. Conclusions To our knowledge, the present study demonstrates for the first time the occurrence of T. callipaeda in mustelids from Romania, records the easternmost locality of the parasite in Europe, and represents the first report of T. callipaeda in the European badger, Meles meles, extending the known host range for this parasite in Europe.

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.

Angiostrongylus chabaudi is a rare cardio-pulmonary nematode infecting felids. Although almost 60 years have passed since the original description of the species in Italy, this parasite has been seldom found in domestic and wildcats in southern Europe. The present study aims to report a new case of patent A. chabaudi infection in a road-killed wildcat from Maramureș County in Northern Romania. The necropsy revealed the presence of parasites in the pulmonary arteries and the right ventricle, and the fecal examination showed the presence of L1 larvae. Parasites were morphologically and morphometrically characterized as A. chabaudi, showing 100 % nucleotide similarity to an Angiostrongylus sp. originating from a wildcat from Germany and 99 % to A. chabaudi from Italy. This study reports A. chabaudi for the first time in Eastern Europe, expanding knowledge about the distribution range of this species.

Ixodes (Afrixodes) aulacodiArthur, 1956 is a poorly known species that has been recorded predominantly in the wet countries of western and central Africa, mainly associated to the greater cane rat Thryonomys swinderianus (Temmink). We herein redescribe the female, describe the male (ascribed to the species from specimens found in copula) and provide the 16S rDNA sequence. We also provide complete illustrations of the adults based on specimens found on greater cane rats in Ivory Coast. Ixodes aulacodi is included in the group of species of the subgenus Afrixodes that have horseshoe shaped anal groove, and which lack auriculae and cornua. The female is easily separated when compared with other species because of a unique combination of characters: All the coxae have internal spurs, coxa II has two external spurs, syncoxae are absent, and trochanters I-III have one spur each. The male has a notched hypostome and lacks syncoxae, auriculae and cornua.

Background A century of debates on the taxonomy of members of the Metastrongyloidea Molin, 1861 led to many reclassifications. Considering the inconstant genus assignation and lack of genetic data, the main aim of this study was to support the validity of the genus Perostrongylus Schlegel, 1934, previously considered a synonym of Aelurostrongylus Cameron, 1927, based on new molecular phylogenetic data and to understand its evolutionary relationships with other metastrongyloid nematodes. Results Specimens of lungworm collected from European badgers in Germany, Romania and Bosnia and Herzegovina were morphologically and molecularly (rDNA, cox1) characterized. From a phylogenetic standpoint, Perostrongylus is grouped with high support together with the genera Filaroides van Beneden, 1858 and Parafilaroides Dougherty, 1946 and includes probably two species: Perostrongylus falciformis (Schlegel, 1933), a parasite of Meles meles in Europe and P. pridhami (Anderson, 1962), a parasite of Neovison vison in North America. Perostrongylus and Aelurostrongylus are assigned to different clades. Aelurostrongylus becomes a monotypic genus, with the only species Aelurostrongylus abstrusus (Railliet, 1898). In addition, we provide morphological and morphometric data for the first-stage (L1), second-stage (L2), and third-stage (L3) larvae of P. falciformis and describe their development in experimentally infected Cornu aspersum snails. The pathological and histopathological lesions in lungs of infected European badgers are also described. This is the first record of P. falciformis in Romania. Conclusions Molecular phylogenetic and morphological data support the validity of the genus Perostrongylus, most probably with two species, P. falciformis in European badgers and P. pridhami in minks in North America. The two genera clearly belong to two different clades: Perostrongylus is grouped together with the genera Filaroides and Parafilaroides (both in the family Filaroididae Schulz, 1951), whereas Aelurostrongylus belongs to a clade with no sister groups.

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.

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.

The golden jackal (Canis aureus) is a species under significant and fast geographic expansion. Various parasites are known from golden jackals across their geographic range, and certain groups can be spread during their expansion, increasing the risk of cross-infection with other carnivores or even humans. The current list of the golden jackal parasites includes 194 species and was compiled on the basis of an extensive literature search published from historical times until April 2017, and is shown herein in synoptic tables followed by critical comments of the various findings. This large variety of parasites is related to the extensive geographic range, territorial mobility and a very unselective diet. The vast majority of these parasites are shared with domestic dogs or cats. The zoonotic potential is the most important aspect of species reported in the golden jackal, some of them, such as Echinococcus spp., hookworms, Toxocara spp., or Trichinella spp., having a great public health impact. Our review brings overwhelming evidence on the importance of Canis aureus as a wild reservoir of human and animal parasites.

Background Angiostrongylus vasorum is the causative agent of canine angiostrongylosis, a severe snail-borne disease of dogs. Red foxes are important natural reservoirs of infection, and surveys of foxes provide a more objective picture of the parasite distribution. Our aim was to investigate the possibility of the presence of A. vasorum in red foxes from the western part of Romania and to analyse the risk factors related to the sex, age and geographic origin of the foxes. Between July 2016 and April 2017, 567 hunted red foxes from 10 counties of western Romania were examined by necropsy for the presence of lungworms. Results Overall, the infection with A. vasorum has been found in 24 red foxes (4.2%) originating in four counties (Mureș, Hunedoara, Sălaj and Cluj). There was no significant difference between the prevalence in males and females, between juveniles and adults and between counties. Conclusions This is the first report of autochthonous infections of A. vasorum in Romania, showing a relatively low prevalence and extending eastwards the known distributional range of this parasite in Europe. The presence of autochthonous cases in domestic dogs in Romania remains to be confirmed by further studies.

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.

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.

Background Thelazia callipaeda is a vector-borne zoonotic nematode parasitizing the conjunctival sac of domestic and wild carnivores, rabbits and humans, with a vast distribution in Asia and the former Soviet Union. In Europe, the nematode has an emerging trend, being reported in Italy, France, Switzerland, Germany, Spain, Portugal, Bosnia and Herzegovina, Croatia, Romania, Greece and Serbia, with human cases known in Italy, France, Spain, Serbia and Croatia. In Romania, the infection was so far reported only in dogs, whereas there are no reports in wildlife despite the large numbers of wild carnivores in the country. The aim of this study was to evaluate the role of wild carnivores in the natural cycle of T. callipaeda in Romania. Methods Between 2014 and 2016, 89 wild carnivores (64 golden jackals, Canis aureus, 13 grey wolves, Canis lupus, nine wildcats, Felis silvestris and three Eurasian lynxes, Lynx lynx) have been examined. During the necropsy, both eyes of all the examined animals have been thoroughly inspected for the presence of parasites. If present, all nematodes were collected in absolute ethanol (for molecular analysis of the partial cox1 gene) or in 4 % formalin (for morphological identification). Results In total, three animals were found to be infected with T. callipaeda: a grey wolf, a golden jackal and a wildcat. The BLAST analysis of all the sequences showed a 100 % similarity to T. callipaeda haplotype h1. To the best of our knowledge, this study represents the first report of T. callipaeda in golden jackals, and the first study on T. callipaeda in wildlife from Romania. Conclusion Our data broaden the host spectrum and geographical distribution of T. callipaeda, highlighting the role of wild carnivores as natural reservoirs for the infection and confirming the ongoing expanding trend of this zoonotic nematode in Europe.

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).

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.

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.

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

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.

Background Hyalomma aegyptium is a hard-tick with a typical three-host life cycle. The main hosts are Palearctic tortoises of genus Testudo. However, other hosts can be used by immature ticks for feeding in natural conditions. Given this complex ecology and multiple host use, the circulation of pathogens by H. aegyptium between various hosts can be important from epidemiological point of view. The aim of this study was to evaluate the role of H. aegyptium as natural carrier of four important zoonotic pathogens. Methods From 2008 to 2011, 448 H. aegyptium ticks were collected from 45 Spur-thighed tortoises, Testudo graeca in Romania. DNA was extracted individually from each tick using a commercial kit. DNA was examined for the presence of specific sequences of Borrelia burgdorferi s.l., Anaplasma phagocytophilum, Ehrlichia canis and Coxiella burnetii by PCR, according to previously described protocols. Results PCR analysis of H. aegyptium revealed the presence of A. phagocytophilum (18.8%), E. canis (14.1%) and C. burnetii (10%). 32.4% of the ticks were infected with at least one pathogen and 9.8% had co-infections. The stages most frequently infected were nymphs (50%) followed by males (33.9%) and females (27%). The number of tortoises which harboured infected ticks was 27/45 examined (60%). From all tested T. graeca, 40% harboured ticks infected with A. phagocytophilum, 46.7% had ticks infected with E. canis and 33.3% had ticks with C. burnetii. This study reports for the first time the presence of A. phagocytophilum and E. canis in H. aegyptium. Conclusions The presence and relatively high prevalence of three important zoonotic pathogens in H. aegyptium raises the question of their epidemiologic importance in disease ecology. As tortoises are unlikely to be reservoir hosts for A. phagocytophilum and E. canis and both these pathogens are common in H. aegyptium, this is an important indication for (1) a possible increased host-switching behaviour of these ticks to competent reservoir hosts (i.e. hedgehogs) and (2) transstadial transmission. Furthermore, if we consider also the presence of C. burnetii, we conclude that T. graeca and its ticks should be evaluated more seriously when assessing the eco-epidemiology of zoonotic diseases.

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.

The overwhelming majority of animal conservation projects are focused on vertebrates, despite most of the species on Earth being invertebrates. Estimates state that about half of all named species of invertebrates are parasitic in at least one stage of their development. The dilemma of viewing parasites as biodiversity or pest has been discussed by several authors. However, ticks were omitted. The latest taxonomic synopses of non-fossil Ixodidae consider valid 700 species. Though, how many of them are still extant is almost impossible to tell, as many of them are known only from type specimens in museums and were never collected since their original description. Moreover, many hosts are endangered and as part of conservation efforts of threatened vertebrates, a common practice is the removal of, and treatment for external parasites, with devastating impact on tick populations. There are several known cases when the host became extinct with subsequent coextinction of their ectoparasites. For our synoptic approach we have used the IUCN status of the host in order to evaluate the status of specifically associated hard-ticks. As a result, we propose a number of 63 coendangered and one extinct hard-tick species. On the other side of the coin, the most important issue regarding tick-host associations is vectorial transmission of microbial pathogens (i.e. viruses, bacteria, protozoans). Tick-borne diseases of threatened vertebrates are sometimes fatal to their hosts. Mortality associated with pathogens acquired from ticks has been documented in several cases, mostly after translocations. Are ticks a real threat to their coendangered host and should they be eliminated? Up to date, there are no reliable proofs that ticks listed by us as coendangered are competent vectors for pathogens of endangered animals.

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.

Wild birds are hosts for several species of ticks, contributing to the maintenance of their local populations in delimited geographic areas. migratory birds play important roles as distributors of ticks within and between distant territories, including continents. Ticks collected from birds are responsible for hosting a significant number of human pathogens. The extensive wetland complex of the Danube Delta provides an internationally important stopover site for millions of birds, belonging to 300 different species, travelling annually. The aim of this study was to detect Anaplasma phagocytophilum in feeding ticks collected from migratory birds along four migratory seasons. Ticks were collected from the birds with forceps and preserved in 96% ethanol for later examination using a separate vial for each bird. A total of 1436 birds in 56 species (15 families) of Passeriformes and 3 non-Passeriformes bird species were captured. A total of 400 ticks were collected and identified as larvae (n = 191; 47.75%), nymphs (n = 201; 50.25%) or adult females (n = 8; 2%). No adult males were found. The ticks belonged to four species (Ixodes ricinus, I. arboricola, I. redikorzevi and Haemaphysalis punctata). Ixodes ricinus was the most common tick (369/400 ticks, 92.25% of the total collected), with a total of 181 larvae, 180 nymphs and 8 females. For detection of Anaplasma phagocytophilum all 400 ticks were examined by PCR, targeting the msp2 gene. Anaplasma phagocytophilum specific DNA was detected in 2 larvae and 2 nymphs of, I. ricinus, from 3 birds prevalence 1.08-%. The infection was absent from the other species of collected ticks The bird species that carried ticks infected with A. phagocytophilum were Turdus merula, Erithacus rubecula and Fringilla coelebs. Several other studies have demonstrated the presence of A. phagocytophilum in ticks collected from birds. Comparing our data with these results, we confirm the low prevalence of A. phagocytophilum, showingthat migratory birds, despite being without reservoir competencemight be important hosts for the dispersal of infected ticks.

Necropsy of 82 specimens of reptiles (snakes, lizards, and turtles) was performed between 2002 and 2006 to determine the helminth fauna. We examined 7 species of reptiles: Natrix natrix (n=25), Natrix tessellata (n=20), Vipera berus (n=5), Coluber caspius (n=12), Anguis fragilis (n=2), Lacerta agilis (n=10), and Emys orbicularis (n=8). Overall prevalence of helminthic infections in Romanian reptiles was 81.71%. In snakes, the most prevalent group of helminths was trematodes. The helminth species, their prevalence, intensity, and abundance are shown in Table 1. In two reptile species, all individuals were infected with helminths (N. natrix and E. orbicularis). The lowest prevalence of helminthic infection was in C. caspius. Three digenean trematodes, namely, Leptophallus nigrovenosus, Metaleptophallus gracillimus, and Spirhapalum polesianum, and three nematodes, Entomelas entomelas, Strongyloides mirzai, and Hexametra quadricornis, are new records for Romania. N. natrix has a new host record for infection with nematode genus Eustrongylides.

Background Dirofilaria immitis and Dirofilaria repens are mosquito-transmitted zoonotic nematodes, causing heartworm disease and skin lesions, respectively, in carnivores. In Europe, the domestic dog is apparently the main definitive host, but patent infections occur also in other species of carnivores. The rapid spread of the golden jackals (Canis aureus) throughout Europe opens a question of involvement of this species in the sylvatic cycle of pathogens in the colonised territories, including Dirofilaria spp. Methods Between January 2014 and May 2015, 54 golden jackals from 18 localities in Romania were examined by full necropsy for the presence of adult filarioid nematodes and blood samples from all animals were screened for the presence of microfilariae of D. immitis, D. repens and Acanthocheilonema reconditum by multiplex PCR DNA amplification. Results Nematodes morphologically identified as D. immitis were found in 18.52 % of the animals, originating from the southern part of Romania. No D. repens or A. reconditum were found at necropsy. The molecular prevalence in blood samples from the same animals was 9.26 % for D. immitis and 1.85 % for D. repens. All samples were negative by PCR for A. reconditum. Conclusion The relatively high prevalence of Dirofilaria spp. infections in golden jackals from Romania together with the increasing density of the jackal populations highlight their potential role in the transmission of these zoonotic parasites and in the maintenance of natural disease foci.

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 Most epidemiological studies on tick-borne pathogens involve collection of ticks from the environment. An efficient collection method is essential for large sample pools. Our main aim was to evaluate the efficacy of a new method, where traditional flagging was enhanced by the use of CO2 dispersed into the white flannel. The CO2 was spread through a rubber hose network inserted into the flag blanket. The research was conducted in spring, in March-April 2011 in two locations from Cluj County, Romania. Methods The research was conducted in March-April 2011 in two locations from Cluj County, Romania. The flag to be tested contained a fine silicone rubber hose network which dispersed the CO2 in the shaft. On each collection site n=30 samplings were performed. Each sampling consisted in the simultaneous use of both flags (with and without CO2) by two persons. The CO2 concentration level on the flag canvas surface was measured. The efficacy of the method was determined by counting comparatively the total number of ticks and separate developmental stage count. Results Using the CO2 improved flag, 2411 (59%) Ixodes ricinus and 100 (53.8%) Dermacentor marginatus ticks were captured, while the CO2-free flag accounted for the collection of 1670 I. ricinus (41%) and 86 (46.2%) D. marginatus ticks. The addition of CO2 prompted a concentration difference on the surface of the flag ranging between 756.5 and 1135.0 ppm with a mean value of 848.9 ppm. Conclusion The study showed that the CO2 enhanced sweep flag increased the ability of I. ricinus (p D. marginatus to be attracted to the flag blanket.

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.