Novel avian oropharyngeal trichomonads isolated from European turtle doves (Streptopelia turtur) and racing pigeons (Columba livia): genetic and morphometric characterisation of clonal cultures.

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.

Tentative identification of the species of Balantidium from ostriches (Struthio camelus) as Balantidium coli-like by analysis of polymorphic DNA.

The characteristics of Balantidium from ostriches (Struthio camelus) are similar to those of Balantidium coli; however, the species Balantidium struthionis was proposed on the basis of the host species (ostriches) and the shape of the macronucleus (with a deep depression in one side). In the present work, we have performed morphological and genetic comparisons between isolates of Balantidium from ostriches and B. coli from pigs to determine the specific status of B. struthionis. The morphological characteristics of the trophozoites of Balantidium from ostriches were reviewed in 100 trophozoites from two isolates. The macronucleus' shape of the ostrich Balantidium was highly variable, thus the use of this criterion for diagnostic purposes is not reliable. Besides, very few trophozoites showed a deep depression in their macronucleus and almost all the trophozoites conform to the description of B. coli. The complete sequence of the DNA coding for the 18s-rRNA-ITS1-5.8s-rRNA-ITS2 regions were obtained by PCR from five pig and five ostrich isolates. The sequences corresponding to the 18s and 5.8s-rRNA genes were identical for the ostrich and pig isolates. Two clearly different genotypes were found in the analysis of the ITS1 and ITS2 regions of the pig isolates; the genotype A was identified in all isolates, while the genotype B was found in only two of them. Their sequences show clear differences from that published corresponding to a B. coli gorilla isolate, which we will consider as a different genotype, C. In our opinion, these different B. coli genotypes reflect the genetic variability of this organism, but further studies would be necessary to determine if it could have practical importance. The polymorphism of the ITS regions have been also found in the ostrich isolates. The same genotypes A and B have been identified, although not as mixed infections. The morphological characteristics and the genetic results suggest that the species name B. struthionis is a synonym of B. coli; however, until experimental infections are carried out to determine if the parasite is transmissible between pigs and ostriches, it would be preferable to tentatively designate it as B. coli-like.

Diterpenoid alkaloids from Delphinium gracile.

Two highly oxygenated hetisine-type diterpenoid alkaloids, delphigraciline (1), 14-hydroxyhetisinone N-oxide (2), and the norditerpenoid alkaloid 8-methoxykarakoline (3), were isolated from a neutral extract of Delphinium gracile. Their structures were elucidated on the basis of spectroscopic data and by comparison with previously reported spectroscopic data of similar alkaloids. Their antiparasitic and insecticidal activities are also discussed.

Entamoeba struthionis n.sp. (Sarcomastigophora: Endamoebidae) from ostriches (Struthio camelus).

In the present work we identify the species of Entamoeba from ostriches (Struthio camelus). The complete sequence of the small subunit ribosomal RNA gene from this organism has been compared with those published for other species of the genus and clear differences have been found. These results confirm previous data which showed differences on parasite morphology and class of host with the other Entamoeba species. Taking all these data together, it can be concluded that the organism from ostriches is a new species whose proposed name is Entamoeba struthionis n.sp. This species probably infects rheas (Rhea americana), but genetic analysis of isolates from this host should be performed to confirm morphological data. Also, comparison of gene sequences with data from other authors on cysts recovered from human stool samples showed the possibility that this amoeba may affect humans. Further studies are needed to determine the risk of transmission of this new species to humans.

Parasites from farmed ostriches (Struthio camelus) and rheas (Rhea americana) in Europe.

During a 4-year-period, more than 500 ostriches and several rheas, all born in European countries and raised in Spain and Portugal, have been analyzed for the presence of ectoparasites and endoparasites. A total of 29 parasite species have been found, most of them of the gastrointestinal tract. Some of the helminth species found may represent spureous parasitosis, as only the eggs (of an ascarid and a trematode) were found in some samples. From the organisms identified, the ectoparasites (lice-Struthiolipeurus rheae, S. nandu; mites-Dermoglyphus pachycnemis, Gabucinia bicaudata), helminths (cestoda-Houttuynia struthionis- and nematoda-Libyostrongylus sp., Codiostomum struthionis-) and the ciliate Balantidium struthionis are known as ratite specific parasites. Capillaria eggs and larvae were also found; there are no previous records of this parasite from ostriches, and the data available do not allow to do a temptative specific diagnosis. Among protozoa, most of the species now found are described for the first time in ratites. They include organisms also found in other birds (Trichomonas gallinae, Tetratrichomonas gallinarum, Chilomastix gallinarum, Spironucleus meleagridis and Pleuromonas jaculans), and organisms whose specific status cannot be established until further analysis are performed (Cryptosporidium sp., Eimeria sp. and/or Isospora sp., Entamoeba sp. of the one-nucleate and of the eight-nucleate mature cyst groups, Endolimax sp., Iodamoeba sp., Monocercomonas sp., Retortamonas sp., Giardia sp., Blastocystis sp. and euglenids).

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.

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.

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.

Entamoeba sp. (Sarcomastigophora: Endamoebidae) from ostriches (Struthio camelus) (Aves: Struthionidae).

The first case of Entamoeba of the 1-nucleate mature cyst group in birds is described. Trophozoites and cysts of Entamoeba have been found in ostriches (Struthio camelus) from farms located all over Spain. The cysts are large (13.47microm mean diameter); they possess one nucleus when mature, with a large endosome and peripheral chromatine arranged in small granules; chromatoid bodies, when present, are large and elongated. Trophozoites are large (19. 88microm mean diameter), with a clear differentiation between ecto- and endoplasm, this containing numerous vacuoles; the nucleus is large and diffuse. The characteristics of this amoeba resembles but do not completely fulfill those of E. suis and E. chattoni; also, these species are from mammals.

In vitro screening of american plant extracts on Trypanosoma cruzi and trichomonas vaginalis.

From the beginning of this decade and with the revival of the phytotherapy, biological research about immunomodulatory, anti-inflammatory and antiprotozoal effects of Central and South American plants have been in progress. Our objective was to determine the antiprotozoal activity of 79 extracts from different plant families, including Asteraceae, Araceae, Moraceae, Solanaceae, Rhamnaceae, Zingiberaceae, Leguminosae and Sapotaceae. Once matching with herbarium specimens authenticated the plants, selected parts were separated, dried carefully and reduced to powder. Most of the screened extracts were aqueous. Two protozoa with different metabolic pathways, Trypanosoma cruzi and Trichomonas vaginalis were used as experimental models. Trypanocidal activity of plants was assayed on epimastigote cultures in liver infusion tryptose (LIT). Anti-Trichomonas activity was determined over cultures of the parasite in Diamond medium. In both cases, microscopic counting of parasites, after their incubation in the presence of different concentrations of the crude extracts, were made in order to determine the cytocidal and cytostatic activities respect to control cultures. Of the nine extracts that showed antiprotozoal activity, those from Mikania cordifolia and Philodendron bipinnatifidum were then fractionated, and again, were assayed the organic and aqueous phases obtained.

Evaluation of drug activity against intracellular forms of Trypanosoma cruzi employing enzyme immunoassay.

OBJECTIVE: To describe application of a new method for the evaluation of anti-Trypanosoma cruzi activity against intracellular forms. METHOD: Vero fibroblasts in 96-well tissue culture plates were infected with trypomastigote forms of T. cruzi. Amastigotes growth was estimated after 24 and 96 h both by microscopic counts of Giemsa-stained monolayers and enzyme-linked immunoIsorbent assay (ELISA). ELISA was performed directly on the fixed cultures using a rabbit anti-T. cruzi immunoIglobulin as the first antibody and a peroxidase-labelled antirabbit immunoglobulin as the second antibody. Three chemical series of structural analogous of gentian violet, thiadiazines and derivatives of 5-nitrothiophene-2-carbaldehyde as well as three reference compounds (nifurtimox, benznidazole and gentian violet) were then assayed. The anti-T. cruzi activity of all of them had been determined previously by microscopic counting of Giemsa-stained infected cultures. RESULTS: None of the assayed compounds showed better activity than the reference ones, but the application of the enzyme immunoassay to quantify the inhibition of growth amastigotes is of great interest, as it yielded results comparable with microscopic counts. CONCLUSION: ELISA can be applied to pharmacological screening, with some advantages over the microscopic examination, including possible automation, rapidity and objectivity in assessment.

Evaluation of a murine model of experimental trichomoniasis.

By using a reference strain of Trichomonas vaginalis and the intraperitoneal route for infecting animals, the influence of the strain of mice, the time observation and the inoculation doses were followed in order to standardize the optimal conditions for the evolution of experimental trichomoniasis. Our results suggest that the inoculation of BALB/c mice with 10(7) trichomonads and the semiquantitative assessment at day 15 postinfection of the gross-pathologic changes in the abdominal cavity--peritoneum, spleen, pancreas, stomach and liver--as well as the presence of ascitic fluid and mortality, maybe a suitable laboratory model of trichomoniasis.

Activity assays of thiadiazine derivatives on Trichomonas vaginalis and amastigote forms of Trypanosoma cruzi.

Eight thiadiazine 1,1-dioxide derivatives were evaluated for antitrichomonal and antitrypanosomal activities. In vivo tests were performed on a murine model for trichomoniasis standardized in our laboratory. The capacity of compounds to clear visceral lesions in experimentally infected animals as well as their effects on the mortality time of mice were used as criteria for activity. One of the thiadiazines (compound 7b) showed an efficacy similar to that obtained with the reference drug metronidazole, although higher doses were required. Its toxicity on cell proliferation in tissue culture was moderate. In vitro assays on amastigote forms of Trypanosoma cruzi were carried out using cultures of Vero cells infected with metacyclic trypomastigotes. For one compound (1) trypanocidal activity resembled that of nifurtimox as assessed by microscopic counts of infected and uninfected cells. Unfortunately, this compound showed a high degree of cytotoxicity on Vero cell cultures.