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.

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.

Genetic identification of the ciliates from greater rheas (Rhea americana) and lesser rheas (Rhea pennata) as Balantioides coli.

The ciliate species Balantioides coli can be cross-transmitted between humans and several animal species. Usually harmless, sometimes it can be pathogenic and cause the death of the host. In birds, B. coli has been confirmed in ostriches by genetic analysis, but the identification from South American greater rheas (Rhea americana) and lesser rheas (Rhea pennata pennata) is tentative. Since these species are reared for commercial purposes and for reintroduction into the wild, it is necessary to elucidate whether the ciliate from rheas is B. coli to minimize health risks for humans and for other domestic and wild animals. Individual parasite cells are collected from Argentinean isolates of reared greater rheas and of wild and reared lesser rheas, and their ITS region was PCR amplified; the cloning products were sequenced and compared with sequences available in public databases. The results have shown that several sequence types are expressed at the same time in the parasite cells, and all correspond to B. coli, confirming the possibility of cross-transmission of the parasite between wild and reared South American rheas and several mammal species and humans.

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.

Trypanocidal, trichomonacidal and cytotoxic components of cultivatedArtemisia absinthium Linnaeus (Asteraceae) essential oil

Artemisia absinthium is an aromatic and medicinal plant of ethnopharmacological interest and it has been widely studied. The use ofA. absinthiumbased on the collection of wild populations can result in variable compositions of the extracts and essential oils (EOs). The aim of this paper is the identification of the active components of the vapour pressure (VP) EO from a selected and cultivated A. absinthiumSpanish population (T2-11) against two parasitic protozoa with different metabolic pathways: Trypanosoma cruzi andTrichomonas vaginalis. VP showed activity on both parasites at the highest concentrations. The chromatographic fractionation of the VP T2-11 resulted in nine fractions (VLC1-9). The chemical composition of the fractions and the antiparasitic effects of fractions and their main compounds suggest that the activity of the VP is related with the presence oftrans-caryophyllene and dihydrochamazulene (main components of fractions VLC1 and VLC2 respectively). Additionally, the cytotoxicity of VP and fractions has been tested on several tumour and no tumour human cell lines. Fractions VLC1 and VLC2 were not cytotoxic against the nontumoural cell line HS5, suggesting selective antiparasitic activity for these two fractions. The VP and fractions inhibited the growth of human tumour cell lines in a dose-dependent manner.

Trypanocidal, trichomonacidal and cytotoxic components of cultivated Artemisia absinthium Linnaeus (Asteraceae) essential oil.

Artemisia absinthium is an aromatic and medicinal plant of ethnopharmacological interest and it has been widely studied. The use ofA. absinthium based on the collection of wild populations can result in variable compositions of the extracts and essential oils (EOs). The aim of this paper is the identification of the active components of the vapour pressure (VP) EO from a selected and cultivated A. absinthium Spanish population (T2-11) against two parasitic protozoa with different metabolic pathways: Trypanosoma cruzi and Trichomonas vaginalis. VP showed activity on both parasites at the highest concentrations. The chromatographic fractionation of the VP T2-11 resulted in nine fractions (VLC1-9). The chemical composition of the fractions and the antiparasitic effects of fractions and their main compounds suggest that the activity of the VP is related with the presence of trans-caryophyllene and dihydrochamazulene (main components of fractions VLC1 and VLC2 respectively). Additionally, the cytotoxicity of VP and fractions has been tested on several tumour and no tumour human cell lines. Fractions VLC1 and VLC2 were not cytotoxic against the nontumoural cell line HS5, suggesting selective antiparasitic activity for these two fractions. The VP and fractions inhibited the growth of human tumour cell lines in a dose-dependent manner.

Trichomonas gypaetinii n. sp., a new trichomonad from the upper gastrointestinal tract of scavenging birds of prey.

In the context of an epidemiological study carried out by several wildlife recovery centers in Spain, trichomonads resembling Trichomonas gallinae were found in the oropharyngeal cavity of 2 Egyptian vultures (Neophron percnopterus) and 14 cinereous vultures (Aegypius monachus) which did not show any symptoms of trichomonosis. In order to characterize them, these isolates along with seven other T. gallinae isolates obtained from different hosts and from different geographical origin were analyzed. Genetic analyses were performed by sequencing the small subunit ribosomal RNA (SSU-rRNA) and the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA regions. The morphological study of the isolates in both light and scanning electron microscopy was also performed. The sequences obtained in the genetic analysis coincide with previously published sequences of an isolate named as Trichomonas sp., obtained from a bearded vulture (Gypaetus barbatus), and showed clear differences to the T. gallinae sequences (97 and 90-91% homology, respectively, for SSU-rRNA and ITS regions) and display higher similarity with Trichomonas vaginalis and Trichomonas stableri than with T. gallinae. Multivariate statistical analysis of the morphometric study also reveals significant differences between the trichomonads of vultures and the isolates of T. gallinae. The isolates from vultures presented smaller values for each variable except for the length of axostyle projection, which was higher. These results together with the different nature of their hosts suggest the possibility of a new species of trichomonad which we hereby name Trichomonas gypaetinii, whose main host are birds of the subfamily Gypaetinae.

Development of a specific polymerase chain reaction assay for the detection of Basidiobolus.

The etiology of chronic diarrhea is complex in humans and animals. It is always necessary to evaluate a list of differential diagnosis, including bacteria, protozoa and fungi. Basidiobolomycosis is a fungal disease reported sporadically worldwide, mainly caused by B. ranarum, a frequent organism found in soil or in the intestine and skin of lizards and frogs. It is an opportunistic pathogen that causes infections characterized by granulomatous lesions in the subcutaneous tissues as well as in the intestinal wall in humans and animals. In this work we have developed a PCR technique to differentiate Basidiobolus from other causes of intestinal disease in dogs and humans. To test the specificity of the PCR assay we included closely related organisms, common intestinal microbiota and pathogenic organisms, such as Aspergillus, Candida, Cryptosporidium, Escherichia, Giardia, Mucor, Proteus, Rhizopus and Salmonella. Pythium insidiosum, which cause clinically similar disease in dogs but require a different treatment. Only Basidiobolus was positive to the PCR assay.

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.