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.

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.

Biological Evaluation of Essential Oils from Selected Medicinal Plants and Their Main Components against Phytomonas davidi (Kinetoplastea: Trypanosomatidae).

Eight essential oils (EOs) from selected medicinal plants have been tested for their activity against Phytomonas davidi, a plant trypanosomal parasite. In the present research, the EOs have been tested on promastigote forms of P. davidi ATCC® 30287™ strain, along with their major components, both separately and in binary combinations, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. The EOs with the highest antipromastigote activity were from Origanum virens and Salvia lavandulifolia. Thymol and ß-pinene were the most active pure compounds. The study of the activity of the pure compounds in combination indicated the existence of antagonistic and synergistic effects depending on the concentration tested. In general, the combinations at low concentrations favored the activity.

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.

Molecular Diversity from Arid-Land Plants: Valorization of Terpenes and Biotransformation Products.

Asteraceae plants from arid lands are a source of biomass, resin and latex rich in terpenoids with diverse biological effects. Thirty-six previously isolated terpenes, comprising sesquiterpenes, diterpenes, triterpenes and quassinoids, isolated from arid-land plants and a series of metabolites from the biotransformation of some lead compounds were evaluated against insect pests (Spodoptera littoralis, Leptinotarsa decemlineata, Myzus persicae and Rhopalosiphum padi), cells (insect, hamster, murine and human tumoral cells) and parasites (Trypanosoma cruzi and Leishmania infantum). Among the insecticidal sesquiterpenes, maalian-1α,8α-diol (12) and γ-eudesmol (17) were antifeedant against L. decemlineata, M. persicae and cytotoxic to Sf9 insect cells, and (-)-maali-3-en-8α-ol (10), (+)-maaliane-5α,8α,9α-triol (11), chrysothame (31) and holacanthone (35) were antifeedant against S. littoralis. The parasite L. infantum was slightly more sensitive than T. cruzi to the test compounds (39 % vs. 33 % of active compounds) with compound 17 and the biotransformed diterpene 27 being antiparasitic to L. infantum, with no cytotoxic effects on mammalian cells. Moreover, sesquiterpenes 3 and 17, and grindelane diterpenes 22, 23 and 26 showed selective activity against chemoresistant human colon, cervical and melanoma cancer cells. Thus, considering our results, the best candidates for future studies are compounds 17 and 3, due to their activity on insect pests, parasites (17) and tumoral cells (3, 17, 22, 23 and 26).

Chemical Composition and Biological Activities of Artemisia pedemontana subsp. assoana Essential Oils and Hydrolate.

Given the importance of the genus Artemisia as a source of valuable natural products, the rare plant Artemisia pedemontana subspecies assoana, endemic to the Iberian Peninsula, has been experimentally cultivated in the greenhouse and aeroponically, to produce biomass for essential oil (EO) extraction. The chemical composition of the EOs was analyzed, and their plant protection (insects: Spodoptera littoralis, Rhopalosiphum padi, and Myzus persicae; plants: Lactuca sativa and Lolium perenne; fungi: Aspergillus niger; and nematode: Meloidogyne javanica) and antiparasitic (Trypanosoma cruzi, Phytomonas davidi, and antiplasmodial by the ferriprotoporphyrin biocrystallization inhibition test) properties were studied, in addition to the hydrolate by-product. The EOs showed a 1,8-cineole and camphor profile, with quantitative and qualitative chemical differences between the cultivation methods. These oils had moderate insect antifeedant, antifungal, and phytotoxic effects; were trypanocidel; and exhibited moderate phytomonacidal effects, while the hydrolate showed a strong nematicidal activity. Both EOs were similarly antifeedant; the EO from the greenhouse plants (flowering stage) was more biocidal (antifungal, nematicidal, and phytotoxic) than the EO from the aeroponic plants (growing stage), which was more antiparasitic. The major components of the oils (1,8-cineole and camphor), or their 1:1 combination, did not explain any of these effects. We can conclude that these EOs have potential applications as insect antifeedants, and as antifungal or antiparasitic agents, depending on the cultivation method, and that the hydrolate byproduct is a potent nematicidal.

Antiparasitic Properties of Cantharidin and the Blister Beetle Berberomeloe majalis (Coleoptera: Meloidae).

Cantharidin (CTD) is a toxic monoterpene produced by blister beetles (Fam. Meloidae) as a chemical defense against predators. Although CTD is highly poisonous to many predator species, some have evolved the ability to feed on poisonous Meloidae, or otherwise beneficially use blister beetles. Great Bustards, Otis tarda, eat CTD-containing Berberomeloe majalis blister beetles, and it has been hypothesized that beetle consumption by these birds reduces parasite load (a case of self-medication). We examined this hypothesis by testing diverse organisms against CTD and extracts of B. majalis hemolymph and bodies. Our results show that all three preparations (CTD and extracts of B. majalis) were toxic to a protozoan (Trichomonas vaginalis), a nematode (Meloidogyne javanica), two insects (Myzus persicae and Rhopalosiphum padi) and a tick (Hyalomma lusitanicum). This not only supports the anti-parasitic hypothesis for beetle consumption, but suggests potential new roles for CTD, under certain conditions.

Trypanocidal Effects of Essential Oils from Selected Medicinal Plants. Synergy among the Main Components.

Fourteen essential oils (EOs) from selected live germplasm of medicinal plants have been tested for their antitrypanosomal and cytotoxic activity. These plants have been domesticated and maintained under experimental cultivation. Their EOs were tested on epimastigote forms of Trypanosoma cruzi strain Y and human lung fibroblasts LC5 cell line, along with the major components of the active oils, both separately and in binary combinations. Mentha rotundifolia, Thymus zygis, T. vulgaris and Hyssopus officinalis were the most active EOs against T. cruzi. Among the main components of these EOs (1-8-cineole, thymol, p-cymene, piperitenone oxide, ß-pinene, γ-terpinene, carvacrol and linalool), the most active against the parasite and less toxic to human cells was thymol. In general, the activity of the main components did not exceed that of their origin EO, and the study of the activity of these compounds in combination indicates the existence of antagonistic and synergistic effects depending on the concentration tested.

Antiparasitic Activity of Diterpenoids Against Trypanosoma cruzi.

Twenty-seven diterpenes, including abietanes, labdanes, abeoabietanes, halimanes, and pimaranes, have been evaluated against epimastigote and intracellular amastigote forms of Trypanosoma cruzi and also against LC5 and NCTC cell lines. Royleanones (3, 4, and 5) and a further abietane (12), obtained by purification of Plectranthus spp. extracts, were the most active compounds on epimastigotes, showing IC50 values similar (1.73 µg/mL, 12) or even lower (0.39, 0.99, and 1.20 µg/mL, 3, 4, and 5 respectively) than the positive control nifurtimox (2.3 µg/mL). On intracellular amastigotes, abietanes 3, 4, and 5 showed a significant activity with IC50 values of 0.83, < 0.31, and 0.62 µg/mL, respectively, but were less potent than the positive control nifurtimox (IC50 < 0.16 µg/mL). Compounds 3, 4, and 5 were not cytotoxic to LC5 and NCTC 929 cells at 1 µg/mL.

New Bioactive Semisynthetic Derivatives of 31-Norlanostenol and Obtusifoliol from Euphorbia officinarum.

Fifteen semisynthetic terpenoid derivatives from the major latex components of Euphorbia officinarum have been evaluated against the insect pest Spodoptera littoralis, two species of protozoan parasites, Trypanosoma cruzi and Leishmania infantum, and also against insect Sf9 and mammalian CHO cells to test their selective cytotoxicity. Our results showed that 40% of the test substances were postingestive toxicants to S. littoralis. All the tested derivatives had cytotoxic effects on insect-derived Sf9 cells, whereas mammalian CHO cells were affected by a lower number of compounds (47%). Furthermore, 87% of the test compounds had antiparasitic effects on both L. infantum and T. cruzi, with some of them being selective parasite toxicants.

Gastrointestinal parasites in greater rheas (Rhea americana) and lesser rheas (Rhea pennata) from Argentina.

Few data exist on the parasites of ratites, especially from regions within their natural range. It is only recently that extensive studies on the parasites of ostriches (Struthio camelus) have been published, mainly from European countries where commercial farming has expanded. Two species of ratites are native in South America: the lesser rhea also known as Darwin's rhea (Rhea pennata) and the greater rhea (Rhea americana). Both species are considered near threatened by the IUCN and are included in the CITES' Appendices I and II, respectively. Parasitological studies have conservation implications, as they allow us to assess the risk of transmission of pathogens from farmed ratites to wild populations. In this study 92 faecal samples from greater rheas and 55 faecal samples from lesser rheas from different localities in Argentine were analyzed to determine their gastrointestinal parasites. In greater rheas the protozoa (Balantidium coli-like and Entamoeba spp.) and helminths (Fasciola hepatica and Deletrocephalus spp.). The protozoa had not previously been cited as parasites of greater rheas in South America. Cysts and/or trophozoites of B. coli-like were found in 16.3% of the samples, while the prevalence of the remaining parasites was below 10%. Lesser rheas harbored the protozoa B. coli-like, Entamoeba spp. and Chilomastix spp. as well as F. hepatica and nematode eggs and larvae. B. coli-like cysts were found in 20.0% of the samples, while the prevalence of the other parasites remained below 5%. Some of them had not been cited as infecting lesser rheas yet.

Antileishmanial, antitrypanosomal, and cytotoxic screening of ethnopharmacologically selected Peruvian plants.

Extracts (34) from eight plant species of the Peruvian Amazonia currently used in traditional Peruvian medicine, mostly as antileishmanial remedies and also as painkiller, antiseptic, antipyretic, anti-inflamatory, antiflu, astringent, diuretic, antipoison, anticancerous, antiparasitic, insecticidal, or healing agents, have been tested for their antileishmanial, antitrypanosomal, and cytotoxic activity. Plant species were selected based on interviews conducted with residents of rural areas. The different plant parts were dried, powdered, and extracted by maceration with different solvents (hexane, chloroform, and 70% ethanol-water). These extracts were tested on promastigote forms of Leishmania infantum strain PB75, epimastigote forms of Trypanosoma cruzi strain Y, and the mammalian CHO cell line. Parasite viability and nonspecific cytotoxicity were analyzed by a modified MTT colorimetric assay method. The isolation and identification of pure compounds from selected extracts were performed by column chromatography, gas chromatography mass spectrometry (GC-MS; mixtures), spectroscopic techniques [MS, infrared (IR), ultraviolet (UV)], and mono and two-dimensional (1)H and (13)C nuclear magnetic resonance (NMR; COSY, HSQC, NOESY) experiments. Chondodendron tomentosum bark and Cedrela odorata were the most active extracts against Leishmania, while C. odorata and Aristoloquia pilosa were the most active against Trypanosoma, followed by Tabebuia serratifolia, Tradescantia zebrina, and Zamia ulei. Six compounds and two mixtures were isolated from Z. ulei [cycasin (1)], T. serratifolia {mixtures 1-2, and naphthoquinones 2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione (2) and 2-(1-hydroxyethyl)-4H,9H-naphtho[2,3-b]furan-4,9-dione (3)}, and C. tomentosum [chondrocurine (4); (S,S')-12-O-methyl(+)-curine (5); and cycleanine (6)]. Four compounds and the two mixtures exhibited significant activity.

Antileishmanial and antitrypanosomal activity of triterpene derivatives from latex of two Euphorbia species.

The in vitro activity on Leishmania infantum promastigotes and Trypanosoma cruzi epimastigotes of 25 semisynthetic terpenoid derivatives has been evaluated. These compounds were obtained through chemical modifications of the major components of Euphorbia resinifera (alpha-euphol and alpha-euphorbol) and Euphorbia officinarum (obtusifoliol and 31-norlanosterol). Leishmaniasis and Chagas' disease are major worldwide health problems. The drugs of choice for their treatment are still problematic in both cases, and therefore there is an urgent need to discover new drugs with high activity and low side effects. Natural products have become a key source of new drugs in the last years. The genus Euphorbia has been the subject of abundant phytochemical and pharmacological research because of its potential medical applications, but the antiparasitic effects of derivatives from plants of this genus are still unknown. Our results showed that 76% and 64% of the test compounds had antiparasitic effects on L. infantum and T cruzi, respectively. The different activities on both parasites, especially their moderate effects on mammalian cells, indicate an interesting selective toxicity.

Genetic heterogeneity in internal transcribed spacer genes of Balantidium coli (Litostomatea, Ciliophora).

The species Balantidium coli is the only ciliate that parasitizes humans. It has been described in other primates, and it has been proposed that the species B. suis from pigs and B. struthionis from ostriches are synonyms of B. coli. Previous genetic analysis of pig and ostrich Balantidium isolates found a genetic polymorphism in the ITS region but its taxonomic relevance was not established. We have extended the genetic analysis to Balantidium isolates of pig, gorilla, human and ostrich origin. We have PCR-amplified and sequenced the ITS region of individual Balantidium cells. The predicted ITS secondary structures of the sequences obtained were transferred by homology modelling to the sequences of other Trichostomatia ciliates (Isotricha, Troglodytella, Lacrymaria and Spathidium) and compared to determine the importance of the differences in the primary sequences. The results show that the ITS2 secondary structure of the species considered follows the general pattern of other ciliates, although with some deviations. There are at least two main types of ITS sequence variants in B. coli which could be present in the same cell and they are common to the mammal and avian hosts studied. These data do not support B. suis and B. struthionis as distinct species.

First report of the genus Retortamonas (Sarcomastigophora: Retortamonadidae) in birds

In studies carried out on the parasites infecting ostriches (Struthio camelus) in Spain, trophozoites of Retortamonas sp. have been found in the intestinal contents of 28 out of 146 slaughtered ostriches. The species infecting ostriches could not be determined from the morphological data available. However, these findings are important as they constitute the first report of the genus Retortamonas in birds

Relationship between biological behaviour and randomly amplified polymorphic DNA profiles of Trypanosoma cruzi strains

Once known some biological characteristics of six Trypanosoma cruzi strains, randomly amplified polymorphic DNA (RAPD) analysis was made. Cluster analysis by UPGMA (unweighted pair group method analysis) was then applied both to biological parameters and RAPD profiles. Inspection of the UPGMA phenograms indicates identical clusters, so supporting that usefulness of biological parameters to characterization of T. cruzi strains still remains

Biological characterization of Trypanosoma cruzi strains

Biological parameters of five Trypanosoma cruzi strains from different sources were determined in order to know the laboratory behaviour of natural populations. The parameters evaluated were growth kinetics of epimastigotes, differentiation into metacyclic forms, infectivity in mammalian cells grown in vitro and parasite susceptibility to nifurtimox, benznidazole and gentian violet. Differences in transformation to metacyclic, in the percentage of infected cells as well as in the number of amastigotes per cell were observed among the strains. Regarding to pharmacological assays, Y strain was the most sensitive to the three assayed compounds. These data demonstrate the heterogeneity of natural populations of T. cruzi, the only responsible of infection in humans