Characterization of Anisotropic Salt Weathering through Nondestructive Techniques Mapping Using a GIS Environment.

Doctrinal texts on architectural heritage conservation emphasize the importance of fully understanding the structural and material characteristics and utilizing information systems. Photogrammetry allows for the generation of detailed, geo-referenced Digital Elevation Models of architectural elements at a low cost, while GIS software enables the addition of layers of material characteristic data to these models, creating different property maps that can be combined through map algebra. This paper presents the results of the mechanical characterization of materials and salt-related decay forms of the polygonal apse of the 13th-century monastery of Santa María de Bonaval (Guadalajara, Spain), which is primarily affected by salt crystallization. Rock strength is estimated using on-site nondestructive testing (ultrasound pulse velocity and Leeb hardness). They are mapped and combined through map algebra to derive a single mechanical soundness index (MSI) to determine whether the decay of the walls could be dependent on the orientation. The presented results show that salt decay in the building is anisotropic, with the south-facing side of the apse displaying an overall lower MSI than the others. The relative overheating of the south-facing side of the apse enhances the effect of salt crystallization, thereby promoting phase transitions between epsomite and hexahydrite.

Bioconsolidation of Damaged Construction Calcarenites and Evaluation of the Improvement in Their Petrophysical and Mechanical Properties.

Bioconsolidation treatment using bacterial carbonatogenesis has been proposed as an environmentally friendly strategy for the efficient preservation of damaged stones, particularly suitable for carbonate stones. The study presented here deals with the evaluation of the performance of this treatment, applied to damaged carbonate stones in two historical buildings in Spain. The methodology applied in this research serves as a reference for future similar studies. Results showed significant improvement in the petrophysical and mechanical properties of the damaged stone following the treatment through the production of calcite and vaterite by the abundant carbonatogenic bacteria inhabiting the stone. These bacteria were able to effectively consolidate weathered areas if an adequate nutritional solution was employed, thereby augmenting the stone's resistance, as evidenced by the Drilling Resistance Measurement System (DRMS). FESEM images showed calcified bacteria and calcified exopolymeric substances (EPS) consolidating stone minerals without blocking their pores. In addition to consolidation, this biotreatment improves the stone's behavior against water absorption and increases the contact angle of water droplets without significant modifications in the pore size or diminishing vapor permeability. No color changes are observed. Overall, these results show that the application of the nutritional solution (M-3P) for in situ consolidation of different types of porous carbonate building stones is a highly effective conservation method, with no modification of the chemical composition of the treated materials.

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.

Integrated post-genomic cell wall analysis reveals floating biofilm formation associated with high expression of flocculins in the pathogen Pichia kudriavzevii.

The pathogenic yeast Pichia kudriavzevii, previously known as Candida krusei, is more distantly related to Candida albicans than clinically relevant CTG-clade Candida species. Its cell wall, a dynamic organelle that is the first point of interaction between pathogen and host, is relatively understudied, and its wall proteome remains unidentified to date. Here, we present an integrated study of the cell wall in P. kudriavzevii. Our comparative genomic studies and experimental data indicate that the general structure of the cell wall in P. kudriavzevii is similar to Saccharomyces cerevisiae and C. albicans and is comprised of ß-1,3-glucan, ß-1,6-glucan, chitin, and mannoproteins. However, some pronounced differences with C. albicans walls were observed, for instance, higher mannan and protein levels and altered protein mannosylation patterns. Further, despite absence of proteins with high sequence similarity to Candida adhesins, protein structure modeling identified eleven proteins related to flocculins/adhesins in S. cerevisiae or C. albicans. To obtain a proteomic comparison of biofilm and planktonic cells, P. kudriavzevii cells were grown to exponential phase and in static 24-h cultures. Interestingly, the 24-h static cultures of P. kudriavzevii yielded formation of floating biofilm (flor) rather than adherence to polystyrene at the bottom. The proteomic analysis of both conditions identified a total of 33 cell wall proteins. In line with a possible role in flor formation, increased abundance of flocculins, in particular Flo110, was observed in the floating biofilm compared to exponential cells. This study is the first to provide a detailed description of the cell wall in P. kudriavzevii including its cell wall proteome, and paves the way for further investigations on the importance of flor formation and flocculins in the pathogenesis of P. kudriavzevii.

Anti-Trypanosomatidae Activity of Essential Oils and Their Main Components from Selected Medicinal Plants.

Kinetoplastida is a group of flagellated protozoa characterized by the presence of a kinetoplast, a structure which is part of a large mitochondria and contains DNA. Parasites of this group include genera such as Leishmania, that cause disease in humans and animals, and Phytomonas, that are capable of infecting plants. Due to the lack of treatments, the low efficacy, or the high toxicity of the employed therapeutic agents there is a need to seek potential alternative treatments. In the present work, the antiparasitic activity on Leishmania infantum and Phytomonas davidi of 23 essential oils (EOs) from plants of the Lamiaceae and Asteraceae families, extracted by hydrodistillation (HD) at laboratory scale and steam distillation (SD) in a pilot plant, were evaluated. The chemical compositions of the EOs were determined by gas chromatography-mass spectrometry. Additionally, the cytotoxic activity on mammalian cells of the major components from the most active EOs was evaluated, and their anti-Phytomonas and anti-Leishmania effects analyzed. L. infantum was more sensitive to the EOs than P. davidi. The EOs with the best anti-kinetoplastid activity were S. montana, T. vulgaris, M. suaveolens, and L. luisieri. Steam distillation increased the linalyl acetate, ß-caryophyllene, and trans-α-necrodyl acetate contents of the EOs, and decreased the amount of borneol and 1,8 cineol. The major active components of the EOs were tested, with thymol being the strongest anti-Phytomonas compound followed by carvacrol. Our study identified potential treatments against kinetoplastids.

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.

Adaptation of the classical end-point ITS-PCR for the diagnosis of avian trichomonosis to a real-time PCR reveals Bonelli's eagle as a new host for Trichomonas gypaetinii.

Avian trichomonosis is a parasitic disease caused mainly by Trichomonas gallinae and other Trichomonas species. It can be asymptomatic, or it can produce a necrotic lesion in the upper digestive tract and spread to other organs, causing the death of the infected birds. In this study, we aimed to evaluate an adapted real-time PCR method for the diagnosis of different genotypes and species of avian oropharyngeal trichomonads. Fifty-six samples from the oropharynx of Bonelli's eagles (Aquila fasciata) obtained between 2018 and 2019 were analyzed using the real-time PCR and the end-point PCR, both targeting trichomonads ITS, and the results were compared by a coefficient of agreement. All positive samples were sequenced. The analysis showed a higher percentage of detection of real-time PCR ITS compared with end-point PCR ITS (64.3 vs 55.4%), and good agreement value (Kappa = 0.816). Melting temperature value for resulting amplicons of real-time PCR for avian trichomonads was 83.45 ± 0.72 °C. Genotypes A, D, and III were found among the sequences. Moreover, Trichomonas gypaetinii, a common species in scavenger birds, is reported for the first time in Bonelli's eagles.

Anti-Trichomonas gallinae activity of essential oils and main compounds from Lamiaceae and Asteraceae plants.

Trichomonas gallinae is a flagellated protozoan that parasitizes the upper digestive tract of various bird species and causes avian trichomonosis. The emergence of resistant strains to the standard treatment, based on nitroimidazoles, increases the need to find alternative therapies. In this study, 36 essential oils (EOs) from Lamiaceae and Asteraceae plant families were tested against T. gallinae trophozoites using the 3-(4,5-dimethylthiazol-2-yl-)-2,5-dipheniltetrazolium bromide (MTT) reduction assay. Among them, EOs from distinct species of Lamiaceae, including the genera Lavandula, Salvia, Thymus, Origanum, and Satureja were the ones reporting better anti-trichomonal activity, and were selected for further analysis, including chemical composition and in vitro assays. The chemical composition of the selected EOs was determined by gas chromatography followed by mass spectrometry and 19 pure compounds were tested against the protozoa, according to their higher abundance in the active EOs. Pure compounds which displayed the highest activity against T. gallinae trophozoites, ordered by highest to lowest activity, were α and ß-thujones, camphene, ß-pinene, linalyl acetate, thymol, 4-terpineol, γ-terpinene, α-pinene, p-cymene, D-fenchone and ß-caryophyllene. A dose dependent effect was observed in most of the EOs and pure compounds tested. The toxicity test conducted in eukaryotic cell cultures with the anti-trichomonal active pure compounds showed that ß-caryophyllene, camphene, α-pinene, and ß-pinene were slightly toxic for Vero cells, and the selectivity index was calculated. Based on the anti-trichomonal activity and the absence of cytotoxicity results, natural products from Lamiaceae plants could be useful as alternative therapy against avian trichomonosis, mainly those containing linalyl acetate, thymol, 4-terpinenol, γ-terpinene, p-cymene and D-fenchone.

Optimization of enzymatic parameters for the production of formononetin from red clover (Trifolium pratense L.) through a response surface methodology.

Trifolium pratense, is a forage found worldwide, but it is negatively impacted by the clover root borer, Hylastinus obscurus. Methanolic extraction has been reported for isolating formononetin from vegetal tissues, with an antifeeding effect on H. obscurus. However, this methodology is time-consuming and also extracts other secondary metabolites, whereas enzymatic assays can provide higher specificity. Hence, the objective of this work was to determine the optimal conditions in pH, temperature, and incubation time for the activity of isoflavone synthase via a response surface model. Once these parameters were optimized, the concentration of formononetin in cultivars and experimental lines of T. pratense was evaluated enzymatically. The results showed that the best condition for developing the enzymatic assay was pH 9.1 with an incubation at 34.5 °C for 155 min. The formononetin content fluctuated between 0.74 and 1.96 mg/g of fresh weight, where Precoz-3, Precoz-1, and Superqueli-INIA presented the highest production.

Characterization of Awp14, A Novel Cluster III Adhesin Identified in a High Biofilm-Forming Candida glabrata Isolate.

Candida glabrata is among the most prevalent causes of candidiasis. Unlike Candida albicans, it is not capable of changing morphology between yeast and hyphal forms but instead has developed other virulence factors. An important feature is its unprecedented large repertoire of predicted cell wall adhesins, which are thought to enable adherence to a variety of surfaces under different conditions. Here, we analyzed the wall proteome of PEU1221, a high biofilm-forming clinical strain isolated from an infected central venous catheter, under biofilm-forming conditions. This isolate shows increased incorporation of putative adhesins, including eight proteins that were not detected in walls of reference strain ATCC 2001, and of which Epa22, Awp14, and Awp2e were identified for the first time. The proteomics data suggest that cluster III adhesin Awp14 is relatively abundant in PEU1221. Phenotypic studies with awp14Δ deletion mutants showed that Awp14 is not responsible for the high biofilm formation of PEU1221 onto polystyrene. However, awp14Δ mutant cells in PEU1221 background showed a slightly diminished binding to chitin and seemed to sediment slightly slower than the parental strain suggesting implication in fungal cell-cell interactions. By structural modeling, we further demonstrate similarity between the ligand-binding domains of cluster III adhesin Awp14 and those of cluster V and VI adhesins. In conclusion, our work confirms the increased incorporation of putative adhesins, such as Awp14, in high biofilm-forming isolates, and contributes to decipher the precise role of these proteins in the establishment of C. glabrata infections.

Editorial for the Special Issue "Parasitic Diseases from Wild Animals with Emphasis on Zoonotic Infections".

The present Editorial intends to briefly describe the findings published in the Special Issue, "Parasitic diseases from wild animals with an emphasis on zoonotic infections". Prevalence data or diagnostic techniques were the focus of several zoonotic parasites transmitted from wildlife, including the protozoa Toxoplasma, Cryptosporidium, Giardia, Blastocystis and Leishmania, and the helminths Echinococcus and Anisakis.

Antiparasitic Effects of Potentially Toxic Beetles (Tenebrionidae and Meloidae) from Steppe Zones.

Arthropods and specifically beetles can synthesize and/or sequester metabolites from dietary sources. In beetle families such as Tenebrionidae and Meloidae, a few studies have reported species with toxic defensive substances and antiparasitic properties that are consumed by birds. Here we have studied the antiparasitic activity of extracts from beetle species present in the habitat of the Great Bustard (Otis tarda) against four pathogen models (Aspergillus niger, Meloidogyne javanica, Hyalomma lusitanicum, and Trichomonas gallinae). The insect species extracted were Tentyria peiroleri, Scaurus uncinus, Blaps lethifera (Tenebrionidae), and Mylabris quadripunctata (Meloidae). M. quadripunctata exhibited potent activity against M. javanica and T. gallinae, while T. peiroleri exhibited moderate antiprotozoal activity. The chemical composition of the insect extracts was studied by gas chromatography coupled with mass spectrometry (GC-MS) analysis. The most abundant compounds in the four beetle extracts were hydrocarbons and fatty acids such as palmitic acid, myristic acid and methyl linoleate, which are characteristic of insect cuticles. The presence of cantharidin (CTD) in the M. quadripunctata meloid and ethyl oleate (EO) in T. peiroleri accounted for the bioactivity of their extracts.

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.

A Systematic Review (1990-2021) of Wild Animals Infected with Zoonotic Leishmania.

Leishmaniasis are neglected diseases caused by several species of Leishmania that affect humans and many domestic and wild animals with a worldwide distribution. The objectives of this review are to identify wild animals naturally infected with zoonotic Leishmania species as well as the organs infected, methods employed for detection and percentage of infection. A literature search starting from 1990 was performed following the PRISMA methodology and 161 reports were included. One hundred and eighty-nine species from ten orders (i.e., Carnivora, Chiroptera, Cingulata, Didelphimorphia, Diprotodontia, Lagomorpha, Eulipotyphla, Pilosa, Primates and Rodentia) were reported to be infected, and a few animals were classified only at the genus level. An exhaustive list of species; diagnostic techniques, including PCR targets; infected organs; number of animals explored and percentage of positives are presented. L. infantum infection was described in 98 wild species and L. (Viania) spp. in 52 wild animals, while L. mexicana, L. amazonensis, L. major and L. tropica were described in fewer than 32 animals each. During the last decade, intense research revealed new hosts within Chiroptera and Lagomorpha. Carnivores and rodents were the most relevant hosts for L. infantum and L. (Viannia) spp., with some species showing lesions, although in most of the studies clinical signs were not reported.

Bacterial Diversity Evolution in Maya Plaster and Stone Following a Bio-Conservation Treatment.

To overcome the limitations of traditional conservation treatments used for protection and consolidation of stone and lime mortars and plasters, mostly based on polymers or alkoxysilanes, a novel treatment based on the activation of indigenous carbonatogenic bacteria has been recently proposed and applied both in the laboratory and in situ. Despite very positive results, little is known regarding its effect on the evolution of the indigenous bacterial communities, specially under hot and humid tropical conditions where proliferation of microorganisms is favored, as it is the case of the Maya area. Here, we studied changes in bacterial diversity of severely degraded tuff stone and lime plaster at the archeological Maya site of Copan (Honduras) after treatment with the patented sterile M-3P nutritional solution. High-throughput sequencing by Illumina MiSeq technology shows significant changes in the bacterial population of the treated stones, enhancing the development of Arthrobacter, Micrococcaceae, Nocardioides, Fictibacillus, and Streptomyces, and, in one case, Rubrobacter (carved stone blocks at Structure 18). In the lime plaster, Arthrobacter, Fictibacillus, Bacillus, Agrococcus, and Microbacterium dominated after treatment. Most of these detected genera have been shown to promote calcium carbonate biomineralization, thus implying that the novel bio-conservation treatment would be effective. Remarkably, the treatment induced the reduction or complete disappearance of deleterious acid-producing bacteria such as Marmoricola or the phylum Acidobacteria. The outcome of this study demonstrates that such a bio-conservation treatment can safely and effectively be applied on temples, sculptures and stuccos of the Maya area and, likely, in other hot and humid environments.

First detection of Leishmania infantum in common urban bats Pipistrellus pipistrellus in Europe.

Leishmania infantum is a protozoan causing leishmaniasis in humans and in dogs, among other animals, which is an endemic disease in the Mediterranean basin. In recent years, the role of wildlife as a possible reservoir of the disease was analyzed and several species of carnivores were reported to have the highest infection rates, with foxes and wolves being the more widely studied species; the role of rabbits and hares as reservoirs of leishmaniasis has also been described. In addition, several studies highlighted the role of bats as suitable hosts for Leishmania species (L. braziliensis, L. major, L. mexicana, and L. tropica) in South and Central America and Africa, but no Leishmania spp. infection in Chiroptera has been reported in Europe. In this study, samples from spleen, hair, and blood were analyzed to detect L. infantum DNA in bats from the Community of Madrid (Spain). Infection by L. infantum was detected in 59.2% of the bats studied (n = 16/27), with the spleen being selected as the site for detection, yielding 14/16 positive results (87.5% sensitivity), followed by hair (n = 7/16) and blood (n = 6/16). In two animals, samples from all three anatomical sites tested positive (7.4% of the total animals), while in four animals the spleen and hair samples tested positive (14.8%), in one animal the blood and hair samples tested positive (3.7%), and in another animal the blood sample only tested positive (3.7%). This is the first report of L. infantum detection in the common urban bat (Pipistrellus pipistrellus) in Europe.

Exploring bacterial community composition in Mediterranean deep-sea sediments and their role in heavy metal accumulation.

The role of microbial processes in bioaccumulation of major and trace elements has been broadly demonstrated. However, microbial communities from marine sediments have been poorly investigated to this regard. In marine environments, particularly under high anthropogenic pressure, heavy metal accumulation increases constantly, which may lead to significant environmental issues. A better knowledge of bacterial diversity and its capability to bioaccumulate metals is essential to face environmental quality assessment. The oligotrophic westernmost Mediterranean, which is highly sensitive to environmental changes and subjected to increasing anthropogenic pressure, was selected for this study. A sediment core spanning the last two millennia was sampled at two intervals, with ages corresponding to 140 (S1) and 1400 (S2) yr BP. High-throughput sequencing showed an abundance of Bacillus, Micrococcus, unclassified members of Planococcaceae, Anaerolineaceae, Planctomycetaceae, Microlunatus, and Microbacterium in both intervals, with slight differences in their abundance, along with newly detected ones in S2, i.e., Propionibacterium, Fictibacillus, Thalassobacillus, and Bacteroides. Canonical correspondence analysis (CCA) and co-occurrence patterns confirmed strong correlations among the taxa and the environmental parameters, suggesting either shared and preferred environmental conditions, or the performance of functions similar to or complementary to each other. These results were further confirmed using culture-dependent methods. The diversity of the culturable bacterial community revealed a predominance of Bacillus, and Micrococcus or Kocuria. The interaction of these bacterial communities with selected heavy metals (Cu, Cr, Zn and Pb) was also investigated, and their capacity of bioaccumulating metals within the cells and/or in the extracellular polymeric substances (EPS) is demonstrated. Interestingly, biomineralization of Pb resulted in the precipitation of Pb phosphates (pyromorphite). Our study supports that remnants of marine bacterial communities can survive in deep-sea sediments over thousands of years. This is extremely important in terms of bioremediation, in particular when considering possible environmentally friendly strategies to bioremediate inorganic contaminants.

Membrane associated proteins of two Trichomonas gallinae clones vary with the virulence.

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.

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.