Occurrence of Leishmania infantum in Wild Mammals Admitted to Recovery Centers in Spain.

Zoonotic leishmaniasis caused by Leishmania infantum is distributed worldwide and affects humans and domestic and wild mammals. In Europe, specifically in the Mediterranean basin, leishmaniasis is endemic due to the concurrence of the phlebotomine vectors and reservoir mammals, including carnivorous wildlife species and other less studied wild species. In this article, spleen, skin, and eye or oral swabs taken from 134 wild mammals admitted to five wildlife recovery centers in Spain were used. PCR employing fragments of the Repeat region, ITS1, and SSUrRNA were used for detection, and positive samples were processed for sequencing. L. infantum was detected in three out of the nine species analyzed, including European hedgehog, European badger, and red squirrel, with percentages ranging from 11.53 to 35.71%, depending on the species. Most of the species showed higher percentages of positivity in spleen samples than in skin samples. A small number of animals from the remaining six species tested negative, including Algerian hedgehog, stone marten, least weasel, garden dormouse, western polecat, and Egyptian mongoose. Hedgehogs and badgers are good candidates for consideration as epidemiological sentinels and pose a higher risk as potential reservoirs of leishmaniasis based on their percentage of infection and wide distribution.

Prevalence of Oestrus ovis in small ruminants from the eastern Iberian Peninsula. A long-term study.

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.

Captive Breeding and Trichomonas gallinae Alter the Oral Microbiome of Bonelli's Eagle Chicks.

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.

Avian Oropharyngeal Trichomonosis: Treatment, Failures and Alternatives, a Systematic Review.

Oropharyngeal avian trichomonosis is a potentially lethal parasitic disease that affects several avian orders. This review is focused on the disease treatments since prophylactic treatment is prohibited in most countries and resistant strains are circulating. A systematic review following the PRISMA procedure was conducted and included 60 articles. Successful and non-toxic treatments of avian oropharyngeal trichomonosis started with enheptin, a drug replaced by dimetridazole, metronidazole, ornidazole, carnidazole and ronidazole. Administration in drinking water was the most employed and recommended method, although hierarchy of the avian flocks and palatability of the medicated water can interfere with the treatments. Besides pigeons, treatments with nitroimidazoles were reported in budgerigars, canaries, finches, bald eagles, a cinereous vulture and several falcon species, but resistant strains were reported mainly in domestic pigeons and budgerigars. Novel treatments include new delivery systems proved with traditional drugs and some plant extracts and its main components. Ethanolic extracts from ginger, curry leaf tree and Dennettia tripetala, alkaloid extracts of Peganum harmala and essential oils of Pelargonium roseum and some Lamiaceae were highly active. Pure active compounds from the above extracts displayed good anti-trichomonal activity, although most studies lack a cytotoxicity or in vivo test.

Prevalence of Toxocara Eggs in Public Parks in the City of Valencia (Eastern Spain).

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.

Invasive Species as Hosts of Zoonotic Infections: The Case of American Mink (Neovison vison) and Leishmania infantum.

Leishmania infantum produces an endemic disease in the Mediterranean Basin that affects humans and domestic and wild mammals, which can act as reservoir or minor host. In this study, we analyzed the presence of the parasite in wild American minks, an invasive species in Spain. We screened for L. infantum DNA by PCR using five primer pairs: Two targeting kinetoplast DNA (kDNA), and the rest targeting the ITS1 region, the small subunit of ribosomal RNA (SSU) and a repetitive sequence (Repeat region). The detection limit was determined for each method using a strain of L. infantum and a bone marrow sample from an infected dog. PCR approaches employing the Repeat region and kDNA (RV1/RV2 primers) showed higher sensitivity than the other PCR methods when control samples were employed. However, only PCR of the Repeat region and nested PCR of SSU (LnSSU) detected the parasite in the samples, while the other three were unable to do so. The majority of the analyzed animals (90.1%) tested positive. American mink may act as an incidental host of the disease for other mammals and should be further investigated, not only for their negative impact on the local fauna, but also as carriers of zoonotic diseases.

Oropharyngeal Trichomonosis Due to Trichomonas gypaetinii in a Cinereous Vulture ( Aegypius monachus) Fledgling in Spain.

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.

RAPD analysis and sequencing of ITS1/5.8S rRNA/ITS2 and Fe-hydrogenase as tools for genetic classification of potentially pathogenic isolates of Trichomonas gallinae.

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.

Occurrence and genetic characterization of Giardia duodenalis from captive nonhuman primates by multi-locus sequence analysis.

Giardia is the most common enteric protozoan that can be pathogenic to both humans and animals. Transmission can be direct through the faecal-oral route, or through ingestion of contaminated water or food. Genetic characterization of Giardia duodenalis isolates has demonstrated the existence of seven groups (assemblages A to G) which differ in their host distribution. Assemblages A and B are present in humans and other primates, dogs, cats, rodents, and other species of wild mammals, but the role of the different host animals in the epidemiology of human infection remains unclear. With this preliminary data, we can infer that nonhuman primates (NHP) might be a potential reservoir for zoonotic transmission. This research paper discusses the presence of Giardia in nonhuman primates housed in two Spanish zoological gardens (located in Valencia and Madrid). Twenty faecal samples obtained from 16 different species of NHP were studied; 70% were positives to Giardia, and genetic analyses were performed by sequencing of four genes (SSrRNA, glutamate dehydrogenase, triose phosphate isomerase, and beta-giardin). The assemblage A was the most frequent (63.4%) in the species studied. A sequence from a red ruffed lemur (corresponding to genotype AI) was obtained, and this is the first reported sequence of a gdh gene obtained from this species. The multi-locus sequence analysis was also performed on the samples positive to nested PCR belonging to assemblage B. After amplification using the GDHeF, GDHiF, and GDHiR gdh primers; AL3543, AL3546, AL3544, and AL3545 tpi primers; G7, G759, GBF, and GBR bg primers, amplicons of 432, 500, and 511 bp respectively were obtained. Amplification products were sequenced and the sequence and phylogenetic analyses showed that genotype IV like was the most frequent in the samples belonging to this assemblage.

Prevalence and genotyping of Trichomonas gallinae in pigeons and birds of prey.

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.