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

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 (Mureş, 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Vector‐borne diseases pose a significant threat to human and animal health worldwide, with arthropods, including fleas and lice, acting as key vectors for transmitting various pathogens. In Uzbekistan, where millions of domestic dogs coexist with humans, the diversity of vector‐borne pathogens carried by ectoparasites remains largely unexplored. This study aimed to investigate the diversity and prevalence of lice and pathogens carried by fleas and lice collected from domestic dogs in Uzbekistan. Ectoparasites were collected from 77 dogs across five regions and subjected to morphological identification and molecular analysis. The 47 lice collected were identified as Trichodectes canis Fabricius, 1780 (Psocodea: Trichodectidae), Linognathus africanus Kellogg and Paine, 1911 (Psocodea: Linognathidae) and Bovicola bovis Linnaeus, 1758 (Phthiraptera: Trichodectidae), alongside with 197 fleas. Notably, L. africanus was detected for the first time on dogs worldwide. Molecular screening detected Rickettsia spp. in 27.9% fleas and 44.7% lice, Dipylidium caninum Linnaeus, 1758 (Cyclophyllidea: Dipylidiidae) in 1.5% of fleas and Bartonella spp. in 0.5% of fleas. Sequencing confirmed Rickettsia asembonensisMaina et al., 2016 (Rickettsiales: Rickettsiaceae), Rickettsia helvetica Beati et al., 1993 (Rickettsiales: Rickettsiaceae), Rickettsia monacensis Simser et al., 2019 (Rickettsiales: Rickettsiaceae), D. caninum and Bartonella clarridgeiae Lawson and Collins, 1996 (Hyphomicrobiales: Bartonellaceae). These findings highlight the potential role of dogs as reservoirs for vector‐borne pathogens and emphasises the need for further research to inform effective disease control strategies. [ABSTRACT FROM AUTHOR]

Subcutaneous dirofilariosis is a fast-spreading infection of dogs, and occasionally of other carnivores and humans. Several factors contribute to its spread, including climate change, which facilitates development and survival of Dirofilaria repens in the mosquito vector. Movement/relocation of infected definitive hosts (dogs) from endemic regions to non-endemic regions is another possible cause of local emergence and the presence of a wide variety of wild reservoirs of the parasite may also contribute to its spread. The main aim of this study was to evaluate the genetic diversity of D. repens from different regions of Europe and to evaluate the spread of identified haplotypes and their geographic origin. A total of 95 D. repens isolates were obtained from Central and Eastern Europe (Poland, Belarus, Ukraine, Austria, Romania), NE Europe (Lithuania, Latvia, Estonia), Italy and Israel. All but two positive samples were obtained from the blood of dogs while one positive sample was obtained from an adult worm from a human case from the Lublin area in SE Poland and one sample was obtained from Anopheles plumbeus mosquito from Austria. Genetic diversity in D. repens isolates was evaluated by PCR amplification and sequencing of three genetic markers, including two mitochondrial genes (mtDNA): the cytochrome c oxidase subunit I (COI) and dehydrogenase subunit I (NADH). Additionally, the genomic marker, internal transcribed spacer 1 (ITS-1) was amplified and sequenced. Haplotypes were differentiated based on sequence alignments by identifying Single Nucleotide Polymorphism (SNPs) using DnaSP and Mega X. PopArt was used to construct a haplotype network including all identified haplotypes. Both mtDNA sequences (COI and NADH) were combined together for phylogenetic and network analyses. Altogether 18 haplotypes (DR1-DR18) were identified in combined mtDNA markers among 95 analysed samples. Haplotype DR1 was the most common encompassing 66 isolates: 42 isolates from Poland (41 from dogs and one from a human), 13 from Lithuania, 4 from Latvia, 2 from Ukraine and 5 from Romania. All other haplotypes grouped around haplotype DR1 separated by 1-5 SNPs, forming a star-like shape. Haplotype DR2 was the second most common haplotype, formed by six isolates from Romania. Interestingly, haplotype DR3 was represented only by four isolates from Israel. The remaining 15 haplotypes were represented by 1-4 isolates of different origins. Our study showed that only minor genetic diversity was found in D. repens since all isolates appear to have clustered in or branched out from haplotype DR1 with 1-5 SNP differences. The genetic diversity appears to be governed by geographic origin since isolates from neighbouring populations (countries) appear to share unique haplotypes while other populations that are geographically distant from individual haplotypes., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Apicomplexan haemoparasites are protozoans that infect a variety of domestic and wild animal species, as well as humans. Data regarding haemoprotozoans in domestic cats are limited; therefore, the aim of this study was to assess the occurrence of Babesia spp., Cytauxzoon spp., and Hepatozoon spp. in domestic cats in Romania using molecular tools., Methods: Blood samples from 371 domestic cats were screened for the presence of piroplasmids. All samples that yielded a visible band in agarose gels were subsequently tested by specific assays targeting the 18S rDNA of Babesia spp., Cytauxzoon spp., and Hepatozoon spp. Moreover, nested PCR assays targeting mitochondrial genes of Babesia spp. were used for screening of all Babesia spp. 18S rDNA-positive samples., Results: From the total number of sampled cats, 19.4% were positive in the PCR assay targeting piroplasmids. Babesia spp. were identified in 15.1% of cats, while 0.5% were positive for Hepatozoon spp. Molecular analyses confirmed the presence of Babesia canis. No samples were positive for Cytauxzoon spp., Conclusions: The high infection rates of domestic cats with Babesia spp. and the need for species differentiation highlight the importance of mitochondrial genes as targets for molecular protocols., (© 2023. The Author(s).)

Background: Equine thelaziosis is a neglected vector-borne parasitic disease in modern veterinary medicine, lacking recent reports. It is transmitted by Musca autumnalis, and potentially other Muscidae species, by ingesting the lachrymal secretions of its equine host. The distribution of both Thelazia lacrymalis and its intermediate hosts remains largely unknown throughout Europe, with most studies dating back 20 years. The aim of this study was to assess the presence, prevalence and distribution of T. lacrymalis in horses from Romania., Methods: The eyes of 273 horses, slaughtered at two abattoirs from the Northwestern and Western regions of Romania, were examined for the presence of T. lacrymalis between March and November 2021. Upon detection, the nematodes were collected and morphologically identified using the keys from literature. Following identification, one specimen from each animal was selected for molecular analysis while the rest underwent detailed morphometric measurements. Mapping and distribution, according to ecoregions, was done using the QGis 3.20 software, while sequences obtained were compared to those available in GenBank through BLAST analysis using the MEGA X software., Results: Of the 273 animals sampled, 12 (4.39%) were positive for Thelazia spp., Infection: Eighty-seven nematodes were recovered, all morphologically identified as T. lacrymalis. The intensity of infestation varied between one and 33 nematodes/animal while five animals presented a bilateral infestation and seven a unilateral one. The highest prevalence was encountered in Pannonian ecoregion (12.12%) while the lowest was in the Alpine ecoregion (0%). Seventy-five intact specimens underwent detailed morphometric analysis, of the 18-20 parameters, resulting in notable differences in striation lengths compared to the data available in other reports. BLAST analysis identified a 96.46-98.60% similarity to the only other COI gene sequence available for T. lacrymalis., Conclusions: The current study represents the first report of T. lacrymalis in horses in Romania. The low prevalence rates are probably linked to the wide use of macrocyclic lactones., (© 2022. The Author(s).)

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., (© 2022. The Author(s).)

Among the zoonotic mosquito-borne nematodes, Dirofilaria repens and Dirofilaria immitis (Spirurida, Onchocercidae) are highly significant from a public health perspective. While D. immitis is also of major veterinary concern, D. repens is regarded as less pathogenic for carnivores, but is the main causative agent of human dirofilariosis throughout the Old World. In the Republic of Uzbekistan, recent data refer exclusively to D. immitis infection in domestic and wild carnivores, while the current prevalence and distribution of D. repens remain unknown. Between 2015 and 2021, a total of 559 domestic and wild carnivore carcasses were collected and examined by necropsy. All subcutaneous nematodes were collected and identified morphologically. The overall prevalence of D. repens infection was of 11.03% in domestic dogs, Canis familiaris, and 9.29% in wildlife hosts: golden jackals, Canis aureus (11.76%), wolves, Canis lupus (9.09%), red foxes, Vulpes vulpes (9.23%), and jungle cats, Felis chaus (7.14%). Additionally, a human case of subcutaneous D. repens infection was also documented. The present study represents the first recent assessment of the occurrence of the zoonotic filarioid D. repens in the Republic of Uzbekistan. It indicates a wide distribution in domestic dogs and four species of wildlife hosts throughout the country, raising awareness on the public health risks associated with this parasite., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Introduction: Rhipicephalus bursa is a common tick parasite of small-to-medium size ungulates, principally in warm, temperate, and subtropical areas. Although common in livestock and showing a wide geographic distribution, its epidemiological role in tick-borne bacterial disease is barely known. This study addressed the knowledge gap and aimed to screen for the presence of Anaplasmataceae and spotted fever group (SFG) Rickettsia species in R. bursa ticks collected from domestic animals in Romania, Eastern Europe., Material and Methods: A total of 64 pools of R. bursa ticks collected from small ungulates were tested by PCR for Anaplasmataceae DNA presence using group-specific primers. Specific testing was performed for Anaplasma marginale/A. centrale/A. ovis , A. platys , A. phagocytophilum , Ehrlichia canis , and SFG Rickettsia . The positive samples were purified and sequenced, and sequences analysis was used to identify the species and to confirm the PCR results., Results: The only pathogen identified in this study was E. canis . The obtained sequences confirmed the PCR results. The presence of E. canis in R. bursa in Romania and in ticks from sheep was shown for the first time in this study., Conclusion: Based on these findings, it may be presumed that the E. canis DNA originated from ticks; however, the vectorial role of R. bursa (and other arthropod species) in the transmission of E. canis should be proved experimentally., Competing Interests: Conflict of Interest Conflict of Interests Statement: The authors declare that there is no conflict of interests regarding the publication of this article., (© 2021 I.A. Matei et al. published by Sciendo.)

The European badger, Meles meles (Carnivora, Mustelidae) is a widespread opportunistic omnivorous mammal. Its food spectrum comprises a wide variety of plants and small animals, occasionally including rodents, such as mice or rats. Considering that rodents are known to play a key role in the life cycle of Trichinella spp., the aims of this study were to investigate the occurrence of these parasites in badgers from Romania and to identify the species. Overall, 61 badgers originating from 14 counties were examined by trichinoscopy and artificial digestion. For species determination, the positive muscle samples, and the larvae recovered from the artificial digestion were used for DNA isolation, and further processed by multiplex PCR. A single badger, originating from Sibiu County, Central Romania, was found positive for Trichinella spp. Five cysts were identified using trichinoscopy: four in the diaphragm and one in the foreleg muscles. Artificial digestion revealed an infection rate of 70 larvae/100 g of muscle. The PCR indicated the occurrence of T. britovi, which is the most commonly detected species in wild carnivores in temperate areas. Although T. britovi has previously been reported in Romania, this represents the first report of its occurrence in the European badger in the country. However, the low prevalence indicates a minor reservoir role of this species., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Protists of the genus Cytauxzoon infect a wide variety of wild and domestic felids worldwide. While the American Cytauxzoon felis has been well described, data on the European isolates of Cytauxzoon are still scant. The aim of the current study was to determine the genetic diversity of European Cytauxzoon spp. in wild felids across Europe by analyzing one nuclear and two mitochondrial genes, along with representative complete mitochondrial genomes. Overall, 106 biological samples from wild felids (92 from Felis silvestris and 14 from Lynx lynx) from Germany, Romania, Czech Republic, and Luxembourg were collected and screened for the presence of Cytauxzoon spp. using nested PCR protocols, targeting the highly conserved 18S rDNA, mitochondrial cytochrome b (CytB) and cytochrome c oxidase subunit I (COI) genes. Furthermore, 18 previously confirmed wild felid biological samples from Europe, and comparative material from USA positive for C. felis, were included in the study. In 18S rDNA sequences analyses, Cytauxzoon spp. from felids formed two separate clades of New World and Old World isolates, with a low inner diversity of the European clade. In contrast to 18S rDNA, the phylogenetic analyses of CytB and COI genes affirmatively revealed three highly supported clades, resulting in three defined genotypes. Similar intra- and interspecific variability of CytB and COI genes was observed in the case of different Babesia spp. Considering geography, host species and analyses of three genes, we conclude that the three detected genotypes of Cytauxzoon in European wild felids represent three new species, which we herein describe., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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

Crows (Corvidae) are common city dwellers worldwide and are increasingly important subjects of epidemiology studies. Although their importance as hosts and transmitters of a number of zoonotic parasites and pathogens is well known, there are no studies on their importance as tick hosts. After mosquitoes, ticks are the most important vectors of zoonotic pathogens, especially for those causing emerging zoonotic diseases. Pathogenic bacteria, especially Borrelia spp., Rickettsia spp., and Anaplasma spp., vectored by ticks, are the cause for most vector-borne diseases in Europe. Here we report on ticks and tick-borne pathogens harbored by urban breeding crows. A total of 36 birds (33.33%, n = 108) hosted ticks, with 91 individual ticks belonging to 6 species (Haemaphysalis concinna, Haemaphysalis parva, Haemaphysalis punctata, Hyalomma marginatum, Ixodes arboricola, and Ixodes ricinus). Rickettsia spp. DNA was found in 6.6% of ticks and 1.9% of bird tissues, whereas Anaplasma phagocytophilum was found in 5.9% of ticks and 0.9% of birds. Two rickettsial genospecies were located, Rickettsia helvetica and Rickettsia monacensis. This is the first study to determine such a diverse tick spectrum feeding on urban corvids, while highlighting their importance as tick hosts and raising concerns about their potential risk to human health.

Thelazia callipaeda is an emerging vector-borne zoonotic helminth parasitizing the conjunctival sac of a broad spectrum of definitive hosts, such as dogs, cats, rabbits, wild carnivores, and humans. Its presence is associated with mild to severe ocular disease. Here, we report two new clinical cases in dogs originating from western and southern Romania, with no travel history. On clinical examination, the nematodes were retrieved from the conjunctival sac and identified using morphological keys and molecular tools. Twenty-two adult nematodes (8 males, 14 females) were collected and were identified as T. callipaeda by morphology. The molecular analysis revealed a 100% identity with haplotype h1 of T. callipaeda. This study describes the occurrence of new autochthonous cases of thelaziosis in Romania, reinforcing the spreading trend of this zoonotic eyeworm and highlighting the need for increased awareness among medical and veterinary practitioners. Moreover, we provide additional molecular evidence for the exclusive distribution of haplotype 1 of T. callipaeda in Europe.