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Dicrocoeliosis is a parasitic disease that mainly affects ruminants during grazing, caused by trematodes of the genus Dicrocoelium, with D. dendriticum being the most common species worldwide. This parasitosis is a chronic and generally subclinical process, with nonspecific signs, which makes its diagnosis challenging. This study aimed to determine the prevalence and seasonal dynamics of D. dendriticum infection in adult sheep from the Valencian Community, eastern Spain, as well as to evaluate the efficacy of flotation and sedimentation techniques when compared with the macroscopic exam of the liver. From February to May 2018, 2019, 2020, 2021, and 2022, a total of 290 adult sheep were examined. The animals were sourced from Castellón province, where a semi-intensive production system predominates. Each animal was euthanized and underwent a macroscopic examination of the liver, as well as a fecal analysis. Among the sampled animals, 117 (40.6%) tested positive for adult trematodes in their liver, while 87 (30%) showed evidence of trematode eggs in the coprological exam, reaching a total of 126 sheep parasitized, with a prevalence of 43.4%. The parasitic burden was established in 90.3 adults per animal when the liver was examined and in 54.5 eggs per gram when the sedimentation coprological exam was performed. No eggs were observed when the flotation technique was employed. A positive correlation was found between the number of adults in the liver and the fecal egg count. No significant differences were detected in the prevalence or parasitic burden throughout the study. Considering the difficulty in controlling the intermediate hosts and the complexity of the life cycle, effective diagnostic methods, combined with the adoption of other preventive measures, is crucial to achieving proper management of this parasitic disease., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Oestrus ovis is an obligate parasite that causes myiasis in domestic ruminants, being commonly found in the Mediterranean area. From 2009 to 2019 a total of 3476 heads of culling sheep and goats from the Mediterranean coast of Spain were examined for the presence of O. ovis. The total prevalence was 56.3%, significantly higher in sheep than in goats (61.2% and 43%, respectively). Differences were found in the mean annual prevalence, with the highest value being registered in 2018 (61.7%) and the lowest in 2012 (50.3%). Autumn, for sheep, and winter, for goats, were the seasons with the highest number of infested specimens. Temperature, but not rainfall, was found to be associated with prevalence (p < 0.05). Most L1 were found in the anatomic region I (septum, meatus, and ventral conchae), while L2 and L3 were mainly located in regions II (nasopharynx, ethmoid labyrinth, and dorsal conchae), and III (sinuses). The overall intensity was 12.8 larvae per head, significantly higher in sheep (13.3) than in goats (3.5). Our results confirm the high prevalence of O. ovis in sheep and goats in this geographic area over the last decade, with the trend increasing in recent years in association with higher mean temperatures., (© 2023 Royal Entomological Society.)

Bonelli's eagle (Aquila fasciata) is an endangered raptor species in Europe, and trichomonosis is one of the menaces affecting chicks at nest. In this paper, we attempt to describe the oral microbiome of Bonelli's eagle nestlings and evaluate the influence of several factors, such as captivity breeding, Trichomonas gallinae infection, and the presence of lesions at the oropharynx. The core oral microbiome of Bonelli's eagle is composed of Firmicutes, Bacteroidota, Fusobacteria and Proteobacteria as the most abundant phyla, and Megamonas and Bacteroides as the most abundant genera. None of the factors analysed showed a significant influence on alfa diversity, but beta diversity was affected for some of them. Captivity breeding exerted a high influence on the composition of the oral microbiome, with significant differences in the four most abundant phyla, with a relative increase of Proteobacteria and a decrease of the other three phyla in comparison with chicks bred at nest. Some genera were more abundant in captivity bred chicks, such as Escherichia-Shigella, Enterococcus, Lactobacillus, Corynebacterium, Clostridium and Staphylococcus, while Bacteroides, Oceanivirga, Peptostreptococcus, Gemella, Veillonella, Mycoplasma, Suttonella, Alloscardovia, Varibaculum and Campylobacter were more abundant in nest raised chicks. T. gallinae infection slightly influenced the composition of the microbiome, but chicks displaying trichomonosis lesions had a higher relative abundance of Bacteroides and Gemella, being the last one an opportunistic pathogen of abscess complications in humans. Raptor's microbiomes are scarcely studied. This is the first study on the factors that influence the oral microbiome of Bonelli's eagle., (© 2022. The Author(s).)

Toxocara spp. is one of the most common zoonotic geohelminths in the world. Its infections are associated with the accidental ingestion of contaminated soil and affecting, especially children. In this study, feces, and soil samples from 14 public parks in the city of Valencia were analyzed. The Telemann method and a modified version of a sieving technique were used to process feces and soil, respectively. None of the fecal samples and 10.9% of soil samples from five parks (35.7%) tested positive for the presence of Toxocara eggs. The most contaminated areas were the canine sanitary parks (30.8% of the samples), followed by socialization areas for dogs (9.7%); no positive samples were found at children's playgrounds. Our results suggest that most pets in Valencia are periodically dewormed, although additional preventive measures should be applied, since the risk of infection exists probably due to the presence of stray dogs and feral cats.

Avian trichomonosis is a parasitic disease that affects wild birds. The objective of this work was to determine the importance of avian trichomonosis in Bonelli's eagles to improve conservation measures in this population. One hundred and eighty-eight birds were studied: 181 chicks, two juveniles, one subadult and four adults. The birds were externally examined and gross lesions at the oropharynx registered. Samples from the oropharyngeal cavity were obtained for Trichomonas spp. detection by culture and PCR, and positive samples were subjected to a multilocus sequence typing approach, including the ITS1/5.8S/ITS2 region (ITS), ribosomal RNA small subunit (18S) and Fe-hydrogenase gene (FeHyd). Global prevalence of T. gallinae infection was 37.8% in total, 45.5% in nestlings. Thirty-three percent of the birds developed lesions that ranged from mild ( n  = 41) to moderate ( n  = 14) or severe ( n  = 7). Multilocus sequence typing analysis showed five different MLS types, ITS-A/18S-VI/FeHyd-A1 and ITS-D/18S-II/Fe-C4 being the most frequent. An association between ITS-A/18S-VI/FeHyd-A1 and moderate or severe lesions was observed, but birds with type ITS-A/18S-VI/FeHyd-A2 also developed lesions. On the contrary, birds with MLS type ITS-D/18S-II/FeHyd-C4 displayed only a low proportion of mild lesions. Chicks raised in nests were at higher risk for T. gallinae infection and development of lesions than chicks raised in captivity. Discordances between samples cultured in TYM and samples subjected to PCR from oropharyngeal swabs were observed, swab-ITS-PCR being more sensitive. RESEARCH HIGHLIGHTS 45.5% of Bonelli's eagles in the nest carried T. gallinae and 39.4% showed lesions.PCR from oral swabs showed higher sensitivity than culture in TYM for detection of T. gallinae .MLS types ITS-A/18S-VI/Fe-A1 (and A2) are a risk factor for the development of lesions.

Thrushes (Turdus spp.) are migratory passerine birds found in northern Europe during the summer months and in southern Europe and north of Africa during the winter. They constitute an important small game bird group very appreciated by Spanish hunters. Between October 2013 and February 2014, 90 adult song thrushes were collected for their exam. After necropsies, three species of helminths were macroscopically recovered from 15 birds (16.7%): Morishitium sp. (16.7%), Splendidofilaria mavis (6.7%) and Dilepis undula (7.8%). One of them showed an adult cestode in the lung. Moreover, 12 of the positive thrushes (80%) harboured microfilaria in pulmonary blood vessels and three of them (20%) were infected by Sarcocystis sp. on skeletal musculature. All parasitized birds showed lesions, ranging from mild to moderate airsacculitis, bronchitis and coelomitis associated to Morishitium sp. infection. To the authors' knowledge, this is the first description of Sarcocystis spp. in song thrushes from Spain. Our results reveal the need for further studies to evaluate the epidemiological role of song thrushes as spreaders of parasites during their annual migration., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Avian trichomonosis is a parasitic disease caused by the flagellated protozoan Trichomonas gallinae. Columbiformes are the reservoir host of the parasite, with high levels of infection, but also other domestic and wild birds from a variety of orders are susceptible to the infection and development of gross lesions. In this paper we describe the type and severity of lesions in wild birds in four avian orders (Accipitriformes, Falconiformes, Strigiformes and Columbiformes). A total of 94 clinical cases diagnosed of trichomonosis were selected for the categorization of their lesions in the upper digestive tract. The affected birds were classified into three different categories (mild, moderate and severe) based on size (in relation to the tracheal opening), depth and location of the lesions. Mild cases are those with small and superficial lesions far from the oropharyngeal opening; moderate cases possess larger and deeper lesions, and severe cases very large and deep lesions that impede swallowing or affect the skull. Mild lesions were found in 10.6 % of cases; moderate lesions were observed in 18.1 % of the birds and severe lesions in 71.3 %. Treatment outcomes in birds with either mild or moderate lesions were favorable, while severe lesions were related to poor body score, leading to death or euthanasia in most cases. A relationship between severe lesions and avian order was found, with a higher percentage of birds with this type in Falconiformes, Columbiformes and Strigiformes. Multifocal lesions were more frequent in Columbiformes and Falconiformes. In Strigiformes, 93.3 % of birds showed lesions in the upper jaw. This study seeks to further understanding of avian trichomonosis and to provide information that will be useful to veterinarians and related professionals for assessment, prognosis and treatment choice for these birds., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Oropharyngeal avian trichomonosis is mainly caused by Trichomonas gallinae, a protozoan parasite that affects the upper digestive tract of birds. Lesions of the disease are characterized by severe inflammation which may result in fatality by starvation. Two genotypes of T. gallinae were found to be widely distributed in different bird species all over the world. Differences in the host distribution and association with lesions of both genotypes have been reported. However, so far no distinct virulence factors of this parasite have been described and studies might suffer from possible co-infections of different genotypes. Therefore, in this paper, we analyzed the virulence capacity of seven clones of the parasite, established by micromanipulation, representing the two most frequent genotypes. Clones of both genotypes caused the maximum score of virulence at day 3 post-inoculation in LMH cells, although significant higher cytopathogenic score was found in ITS-OBT-Tg-1 genotype clones at days 1 and 2, as compared to clones with ITS-OBT-Tg-2. By using one representative clone of each genotype, a comparative proteomic analysis of the membrane proteins enriched fraction has been carried out by a label free approach (Data available via ProteomeXchange: PXD013115). The analysis resulted in 302 proteins of varying abundance. In the clone with the highest initial virulence, proteins related to cell adhesion, such as an immuno-dominant variable surface antigen, a GP63-like protein, an armadillo/beta-catenin-like repeat protein were found more abundant. Additionally, Ras superfamily proteins and calmodulins were more abundant, which might be related to an increased activity in the cytoskeleton re-organization. On the contrary, in the clone with the lowest initial virulence, larger numbers of the identified proteins were related to the carbohydrate metabolism. The results of the present work deliver substantial differences between both clones that could be related to feeding processes and morphological changes, similarly to the closely related pathogen Trichomonas vaginalis., Competing Interests: The authors have declared that no competing interests exist.

A juvenile Cinereous Vulture ( Aegypius monachus) fledgling was found disorientated on the roof of a building in Madrid City, Spain, in October 2016. A veterinary examination revealed multiple plaques distributed throughout the oropharyngeal cavity. Lesions were located under the tongue and at the choanal slit, hard palate, and esophagus opening and ranged from 2 to 7 mm, coalescing in areas up to 2 cm, with a yellowish color of the surface. Motile trichomonad trophozoites were detected in fresh wet mount smears from the lesions. Sequence analysis of the internal transcribed spacer (ITS)1/5.8S/ITS2 and small subunit ribosomal RNA confirmed that Trichomonas gypaetinii was the etiologic agent. Microbiologic cultures did not reveal any pathogenic bacteria or fungi. The animal recovered successfully after treatment with metronidazole and trimethoprim-sulfamethoxazole and was later released in a suitable habitat. Avian trichomonosis lesions caused by T. gypaetinii have not been reported.

Extensive diversity has been described within the avian oropharyngeal trichomonad complex in recent years. In this study we developed clonal cultures from four isolates selected by their different ITS1/5.8S/ITS2 (ITS) genotype and their association with gross lesions of avian trichomonosis. Isolates were obtained from an adult racing pigeon and a nestling of Eurasian eagle owl with macroscopic lesions, and from a juvenile wood pigeon and an European turtle dove without clinical signs. Multi-locus sequence typing analysis of the ITS, small subunit of ribosomal rRNA (SSUrRNA) and Fe-hydrogenase (Fe-hyd) genes together with a morphological study by optical and scanning electron microscopy was performed. No significant differences in the structures were observed with scanning electron microscopy. However, the genetic characterisation revealed novel sequence types for the SSUrRNA region and Fe-hyd gene. Two clones were identified as Trichomonas gallinae in the MLST analysis, but the clones from the racing pigeon and European turtle dove showed higher similarity with Trichomonas tenax and Trichomonas canistomae than with T. gallinae at their ITS region, respectively. SSUrRNA sequences grouped all the clones in a clade that includes T. gallinae, T. tenax and T. canistomae. Further diversity was detected within the Fe-hyd locus, with a clear separation from T. gallinae of the clones obtained from the racing pigeon and the European turtle dove. In addition, morphometric comparison by optical microscopy with clonal cultures of T. gallinae revealed significant statistical differences on axostyle projection length in the clone from the European turtle dove. Morphometric and genetic data indicate that possible new species within the Trichomonas genus were detected. Taking in consideration the diversity in Trichomonas species present in the oral cavity of birds, a proper genetic analysis is highly recommended when outbreaks occur., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Many obligate and facultative avian scavengers are increasingly dependent on food provided in supplementary feeding stations (SFS), which are managed for the conservation of these species. Deliberate feeding can influence disease-related host demography and population dynamics through physiological changes and density-dependent parasite acquisition and transmission, but information on this threat to avian scavengers is scarce. Due to their effects on host aggregation and density, we hypothesised that the predictability and concentration of food in SFS can exacerbate parasite infection. This hypothesis was tested by comparing the prevalence, richness, abundance and mixed infection of endoparasites (coccidia and helminths) in red kites Milvus milvus foraging on livestock carcasses (mostly of pigs and poultry) in overcrowded and confined conditions at SFS, relative to those foraging alone or in small groups on wild prey unevenly randomly distributed within large areas during winter, mostly wild rabbits (Oryctolagus cuniculus). No clear differences were found between areas with and without SFS in the prevalence and abundance of oocyst of Eimeria. This coccidian genus appears to include parasites of the prey rather than the raptors, thus representing parasite transport or pseudo-parasitism rather than actual parasitism in the kites. A higher prevalence and richness of helminths, as well as mixed infections with several phyla, was found in kites exploiting SFS than in those feeding on wild prey in the area without SFS. The unsanitary conditions derived from the stack of livestock carcasses and the contamination of carrion with the faeces of multiple scavenger hosts can increase the accumulation and persistence of helminths eggs and intermediate hosts. The regular use and frequent confinement of large numbers of red kites at SFS can promote the spread of parasites to a large proportion of the European breeding population distributed across Spain during the winter. We encourage that carcasses of free roaming livestock can be left in the countryside, as well as the conservation management of wildlife exploited as food by red kites (especially wild rabbits), to attempt avoiding overcrowded and confined conditions at SFS. Further research is required to assess the impact of deliberate feeding on the spread of parasites and other disease agents in the threatened species SFS are intended to favour., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Trichomonas gallinae is a worldwide parasite that causes oropharyngeal avian trichomonosis. During eight years, 60 axenic isolates were obtained from different bird species and characterized by three molecular methods: RAPD analysis and PCR-sequencing of ITS1/5.8S rRNA/ITS2 fragment and Fe-hydrogenase gene. We have found two genotypes of ITS1/5.8S rRNA/ITS2 widely distributed among bird populations, a new variant and also two sequences with mixed pattern. Genotype ITS-OBT-Tg-1 was associated with the presence of gross lesions in birds. We have found eight genotypes of the Fe-hydrogenase (A1, A2, C2, C2.1, C4, C5, C6 and C7), three of them are new reports (C5, C6 and C7), and also three sequences with mixed pattern. Subtype A1 of the Fe-hydrogenase was also related with the presence of lesions. RAPD analyses included most of the strains isolated from animals with lesions in one of the sub-clusters. Potentially pathogenic isolates of T. gallinae obtained in this study fulfill the following criteria with one exception: isolated from lesions+ITS-OBT-Tg-1 genotype+FeHyd A1+RAPD sub-cluster I2., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Oropharyngeal trichomonad isolates of wild birds from Spain were studied. A total of 1688 samples (1214 of predator birds and 474 of prey species) from wildlife recovery centres and scientific bird-ringing campaigns were analysed from 2011 to 2013. The overall infection prevalence was 20.3% (11.4% in predator birds and 43.3% in prey species). Pathognomonic lesions were present in 26% of the infected birds (57.3% in predator birds and 4.9% in prey species). The most commonly parasitized species were the goshawk (Accipiter gentilis, 74.5%) and the rock pigeon (Columba livia, 79.4%). Host species in which the parasite has not been previously analysed by polymerase chain reaction and sequencing in Spain are also reported: Columba palumbus, Streptopelia turtur, Pica pica, A. gentilis, Accipiter nisus, Asio otus, Bubo bubo, Buteo buteo, Circus aeruginosus, Circus cyaneus, Falco naumanni, Falco peregrinus, Neophron percnopterus, Otus scops, Pernis apivorus and Strix aluco. Sequence analysis of the ITS1/5.8S/ITS2 region revealed five different genotypes and also some mixed infections. A relationship between genotype and host species was observed, but only two genotypes (ITS-OBT-Tg-1and ITS-OBT-Tg-2) were widely distributed. Genotype ITS-OBT-Tg-1 was most frequently found in predator birds and statistically associated with pathognomonic lesions. Non-strict ornithophagous species were at higher risk to develop disease than ornithophagous ones. Genotypes ITS-OBT-Tcl-1 and ITS-OBT-Tcl-2 are new reports, and ITS-OBT-Tvl-5 is reported for the first time in Spain. They showed higher genetic homology to Trichomonas canistomae and Trichomonas vaginalis than to Trichomonas gallinae, indicating the possibility of new species within this genus.

Three hundred and eighty six faecal specimens were randomly collected from 1- to 3-month-old lambs from 16 farms in Spain to investigate the presence of different genotypes of Giardia duodenalis. Individual specimens were examined by IFA (Immunofluorescence assay) and beta-giardin PCR polymerase chain reaction. Cysts of G. duodenalis were shed by lambs in every flock analyzed, showing a prevalence by farms of 100%. The average prevalence of G. duodenalis for the 386 specimens was 42%, ranging from 8.3 to 80% depending on the farm. beta-giardin PCR positive samples were sequenced to determine the genotypes present at each farm and seven new subtypes of beta-giardin Assemblage E are reported in this study. In each farm, one to six different beta-giardin subtypes were found, showing the high variability of the target. Also, one flock had the zoonotic Assemblage A. This is the first report of Giardia subgenotype A-1 in sheep in Spain.

Avian trichomonosis is a world-wide parasitic disease caused by the protozoan Trichomonas gallinae. Although several degrees of pathogenicity have been described on the basis of the clinical signs in birds, there are few reports concerning the genetic characterization of the parasite and its relationship with pathogenicity. The parasite usually appears apathogenic but is occasionally responsible for outbreaks of the disease in avian populations, particularly affecting nestlings of ornithophagous raptors. For 3 years, cultures of oropharingeal samples from 612 wild and domestic pigeons (Columba livia) and 102 birds of prey from 15 different species were made in an attempt to determine the prevalence of T. gallinae in the Valencian Community (eastern Spain). To establish the genotype of the isolates, 5.8S rRNA and the surrounding internal transcribed spacer regions were amplified by polymerase chain reaction and were sequenced. After restriction map analysis, sequencing and polymerase chain reaction-restriction fragment length polymorphism using HaeIII showed two genotypes (A and B) in isolates from both groups of birds, although genotype prevalence differed in each group-genotype A being more prevalent in columbiforms and genotype B in raptors. In addition, genotype B was present in every bird that displayed macroscopic lesions.

Three hundred and ninety-five pig fecal samples were analyzed looking for Blastocystis sp. using optical microscopy and PCR. A global prevalence of 46.8% has been observed in this study, although relative values of prevalence differ notably according to the strata examined, ranging from 9.3% in sows to 75% in weaners. Statistic analysis of the data included several risk factors such as different management systems, date of sample collection, fecal consistency, age and sex of the animals. The presence of the parasite was statistically associated to the variables "age" and "date of sample collection", being more prevalent in weaners and grower pigs and warm season. Random fragment-length polymorphism (RFLP-PCR) analysis of positive PCR samples revealed a high homology in the digestion pattern, appearing as two ribotypes. The results were further confirmed by sequencing of ten randomly selected samples, showing that the samples obtained in this study were included in two genotypes: genotype I previously named by Noël et al. [Noël, C., Dufernez, F., Gerbod, D., Edgcomb, V.P., Delgado-Viscogliosi, P., Ho, L.-Ch., Singh, M., Wintjens, R., Sogin, M.L., Capron, M., Pierce, R., Zenner, L., Viscogliosi, E., 2005. Molecular phylogenies of Blastocystis isolates from different hosts: implications for genetic diversity, identification of species, and zoonosis. J. Clin. Microbiol. 43, 348-355], in which Blastocystis sp. sequences from humans, pigs and cattle were included, and genotype II, which only included Blastocystis hominis sequences obtained from human and other primates. This is the first report including Blastocystis sequences from swine origin in genotype II.