Pan-American Trypanosoma (Megatrypanum) trinaperronei n. sp. in the white-tailed deer Odocoileus virginianus Zimmermann and its deer ked Lipoptena mazamae Rondani, 1878: morphological, developmental and phylogeographical characterisation.

BACKGROUND: The subgenus Megatrypanum Hoare, 1964 of Trypanosoma Gruby, 1843 comprises trypanosomes of cervids and bovids from around the world. Here, the white-tailed deer Odocoileus virginianus (Zimmermann) and its ectoparasite, the deer ked Lipoptena mazamae Rondani, 1878 (hippoboscid fly), were surveyed for trypanosomes in Venezuela. RESULTS: Haemoculturing unveiled 20% infected WTD, while 47% (7/15) of blood samples and 38% (11/29) of ked guts tested positive for the Megatrypanum-specific TthCATL-PCR. CATL and SSU rRNA sequences uncovered a single species of trypanosome. Phylogeny based on SSU rRNA and gGAPDH sequences tightly cluster WTD trypanosomes from Venezuela and the USA, which were strongly supported as geographical variants of the herein described Trypanosoma (Megatrypanum) trinaperronei n. sp. In our analyses, the new species was closest to Trypanosoma sp. D30 from fallow deer (Germany), both nested into TthII alongside other trypanosomes from cervids (North American elk and European fallow, red and sika deer), and bovids (cattle, antelopes and sheep). Insights into the life-cycle of T. trinaperronei n. sp. were obtained from early haemocultures of deer blood and co-culture with mammalian and insect cells showing flagellates resembling Megatrypanum trypanosomes previously reported in deer blood, and deer ked guts. For the first time, a trypanosome from a cervid was cultured and phylogenetically and morphologically (light and electron microscopy) characterised. CONCLUSIONS: In the analyses based on SSU rRNA, gGAPDH, CATL and ITS rDNA sequences, neither cervids nor bovids trypanosomes were monophyletic but intertwined within TthI and TthII major phylogenetic lineages. One host species can harbour more than one species/genotype of trypanosome, but each trypanosome species/genotype was found in a single host species or in phylogenetically closely related hosts. Molecular evidence that L. mazamae may transmit T. trinaperronei n. sp. suggests important evolutionary constraints making tight the tripartite T. trinaperronei-WTD-deer ked association. In a plausible evolutionary scenario, T. trinaperronei n. sp. entered South America with North American white-tailed deer at the Pliocene-Pleistocene boundary following the closure of the Panama Isthmus.

Nucleolar Structure and Function in Trypanosomatid Protozoa.

The nucleolus is the conspicuous nuclear body where ribosomal RNA genes are transcribed by RNA polymerase I, pre-ribosomal RNA is processed, and ribosomal subunits are assembled. Other important functions have been attributed to the nucleolus over the years. Here we review the current knowledge about the structure and function of the nucleolus in the trypanosomatid parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania ssp., which represent one of the earliest branching lineages among the eukaryotes. These protozoan parasites present a single nucleolus that is preserved throughout the closed nuclear division, and that seems to lack fibrillar centers. Trypanosomatids possess a relatively low number of rRNA genes, which encode rRNA molecules that contain large expansion segments, including several that are trypanosomatid-specific. Notably, the large subunit rRNA (28S-type) is fragmented into two large and four small rRNA species. Hence, compared to other organisms, the rRNA primary transcript requires additional processing steps in trypanosomatids. Accordingly, this group of parasites contains the highest number ever reported of snoRNAs that participate in rRNA processing. The number of modified rRNA nucleotides in trypanosomatids is also higher than in other organisms. Regarding the structure and biogenesis of the ribosomes, recent cryo-electron microscopy analyses have revealed several trypanosomatid-specific features that are discussed here. Additional functions of the nucleolus in trypanosomatids are also reviewed.

Antiparasitic effect of Dinoponera quadriceps giant ant venom.

Neglected tropical diseases (NTD) are treated with toxic therapy of limited efficacy. Previously, we studied the antimicrobial effect of Dinoponera quadriceps venom (DqV) against bacteria. To continue the study, we report in this short communication the antimicrobial effect of DqV against Leishmania amazonensis and Trypanosoma cruzi. DqV inhibits the promastigote forms of L. amazonensis and all T. cruzi developmental forms, with low toxicity in host cells. DqV causes cell death in T. cruzi through necrotic and apoptotic mechanisms observed by staining the cells with annexin V-FITC (AX) and propidium iodide (PI), loss of mitochondrial membrane potential by flow cytometry analyses and confocal microscopy and morphological alterations, such as loss of membrane integrity and cell shrinkage by scanning electron microscopy (SEM). In conclusion, we suggest there is an antimicrobial effect also on parasites.

Developmental and Ultrastructural Characterization and Phylogenetic Analysis of Trypanosoma herthameyeri n. sp. of Brazilian Leptodactilydae Frogs.

We described the phylogenetic affiliation, development in cultures and ultrastructural features of a trypanosome of Leptodacylus chaquensis from the Pantanal biome of Brazil. In the inferred phylogeny, this trypanosome nested into the Anura clade of the basal Aquatic clade of Trypanosoma, but was separate from all known species within this clade. This finding enabled us to describe it as Trypanosoma herthameyeri n. sp., which also infects other Leptodacylus species from the Pantanal and Caatinga biomes. Trypanosoma herthameyeri multiplies as small rounded forms clumped together and evolving into multiple-fission forms and rosettes of epimastigotes released as long forms with long flagella; scarce trypomastigotes and glove-like forms are common in stationary-phase cultures. For the first time, a trypanosome from an amphibian was observed by field emission scanning electron microscopy, revealing a cytostome opening, well-developed flagellar lamella, and many grooves in pumpkin-like forms. Transmission electron microscopy showed highly developed Golgi complexes, relaxed catenation of KDNA, and a rich set of spongiome tubules in a regular parallel arrangement to the flagellar pocket as confirmed by electron tomography. Considering the basal position in the phylogenetic tree, developmental and ultrastructural data of T. herthameyeri are valuable for evolutionary studies of trypanosome architecture and cell biology.

Aflagellar epimastigote forms are found in axenic culture of Trypanosoma caninum.

Representatives of the genus Trypanosoma have been traditionally found in epimastigote, espheromastigote and trypomastigote flagellated forms in axenic cultures. Trypanosoma caninum is a trypanosomatid that has recently been reported infecting dogs in endemic areas of canine leishmaniasis in Brazil. It presents specific biological characteristics and it is found exclusively on healthy skin. Here, we describe the evolutive forms of this parasite showing not only the forms commonly found in culture, but also epimastigote forms with no free flagellum. The study was conducted using scanning and transmission electron microscopy and, we demonstrate that typical flagellated epimastigotes originate from forms without flagellum, although the latter may remain without differentiation in the culture. Two hypotheses are considered and discussed in this paper: (i) the aflagellated epimastigotes are a typical developmental forms of T. caninum and (ii) the emergence of these aflagellated forms could be resultant from a disturbed process during cell division caused by interfering specific proteins, which leads to inability to form and regulate the flagellum length. In any case, considering that T. caninum is a parasite that is still little studied, the information brought by our study adds data which may be useful to clarify aspects on the cell cycle of this intriguing parasite that has been found in different regions of Brazil.

Isolation and in vitro maintenance of trypanosomes from naturally infected and commercially important Brazilian fish.

Fish trypanosomes are widely distributed in commercially important fish, with high prevalence in some Brazilian species. This study provides the first record of the isolation and in vitro maintenance of trypanosomes from Brazilian fish. We produced 49 trypanosome isolates from naturally infected catfish (Hypostomus affinis and Hypostomus luetkeni), using 9 different culture media (out of 31 tested). Trypanosomes were maintained in culture for at least 15 mo and were successfully cryopreserved. Culture forms-epimastigotes and short trypomastigotes-were capable of dividing in vitro. Our study is an important step in the investigation of ultrastructure, taxonomy, and phylogeny of trypanosomes from commercially important Brazilian fish.

Morphological and molecular characterization and phylogenetic relationships of a new species of trypanosome in Tapirus terrestris (lowland tapir), Trypanosoma terrestris sp. nov., from Atlantic Rainforest of southeastern Brazil.

BACKGROUND: The Lowland tapir (Tapirus terrestris) is the largest Brazilian mammal and despite being distributed in various Brazilian biomes, it is seriously endangered in the Atlantic Rainforest. These hosts were never evaluated for the presence of Trypanosoma parasites. METHODS: The Lowland tapirs were captured in the Brazilian southeastern Atlantic Rainforest, Espírito Santo state. Trypanosomes were isolated by hemoculture, and the molecular phylogeny based on small subunit rDNA (SSU rDNA) and glycosomal-3-phosphate dehydrogenase (gGAPDH) gene sequences and the ultrastructural features seen via light microscopy and scanning and transmission electron microscopy are described. RESULTS: Phylogenetic trees using combined SSU rDNA and gGAPDH data sets clustered the trypanosomes of Lowland tapirs, which were highly divergent from other trypanosome species. The phylogenetic position and morphological discontinuities, mainly in epimastigote culture forms, made it possible to classify the trypanosomes from Lowland tapirs as a separate species. CONCLUSIONS: The isolated trypanosomes from Tapirus terrestris are a new species, Trypanosoma terrestris sp. n., and were positioned in a new Trypanosoma clade, named T. terrestris clade.

Phylogenetic analyses based on small subunit rRNA and glycosomal glyceraldehyde-3-phosphate dehydrogenase genes and ultrastructural characterization of two snake Trypanosomes: Trypanosoma serpentis n. sp. from Pseudoboa nigra and Trypanosoma cascavelli from Crotalus durissus terrificus.

We sequenced the small subunit (SSU) rRNA and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) genes of two trypanosomes isolated from the Brazilian snakes Pseudoboa nigra and Crotalus durissus terrificus. Trypanosomes were cultured and their morphometrical and ultrastructural features were characterized by light microscopy and scanning and transmission electron microscopy. Phylogenetic trees inferred using independent or combined SSU rRNA and gGAPDH data sets always clustered the snake trypanosomes together in a clade closest to lizard trypanosomes, forming a strongly supported monophyletic assemblage (i.e. lizard-snake clade). The positioning in the phylogenetic trees and the barcoding based on the variable V7-V8 region of the SSU rRNA, which showed high sequence divergences, allowed us to classify the isolates from distinct snake species as separate species. The isolate from P. nigra is described as a new species, Trypanosoma serpentis n. sp., whereas the isolate from C. d. terrificus is redescribed here as Trypanosoma cascavelli.

Trypanosoma (Megatrypanum) saloboense n. sp. (Kinetoplastida: Trypanosomatidae) parasite of Monodelphis emiliae (Marsupiala: Didelphidae) from Amazonian Brazil.

Trypanosoma (Megatrypanum) soloboense n. sp., is described in the Brazilian opossum Monodelphis emiliae (Thomas, 1912) from primary forest in the Salobo area of the Serra dos Carajás (6 degrees S, 50 degrees 18' W) Pará State, North Brazil. Two morphologically different trypomastigotes were noted. Slender forms, regarded as immature parasites, have a poorly developed undulating membrane adhering closely to the body: large, broad forms with a well developed membrane are considered to be the mature trypomastigotes and have a mean total length of 71.2 microm (62.4-76.2) and a width of 6.1 (5.0-8.0). Infections studied in two opossums were of very low parasitaemia. The large size of T. (M.) saloboense readily distinguishes it from the two previously described members of the subgenus Megatrypanum of neotropical marsupials, T. (M.) freitasi Régo et al., 1957 of Didelphis ozarae and D. marsupialis, and T. (M.) samueli Mello, 1977 of Monodelphis domesticus, which measure only 49.0-51.5 microm and 42.4 microm respectively. No infections were obtained in hamsters inoculated with triturated liver and spleen from one infected M. emiliae, or in laboratory mice inoculated with epimastigotes from a blood-agar culture. No division stages could be detected in the internal organs or the peripheral blood.

First record of Trypanosoma chattoni in Brazil and occurrence of other Trypanosoma species in Brazilian frogs (Anura, Leptodactylidae).

The present study provides the first record of Trypanosoma chattoni Mathis and Leger, 1911, in a new host, Leptodactylus fuscus Schneider, 1799 (Anura, Leptodactylidae), and the occurrence of Trypanosoma rotatorium-like species in Leptodactylus chaquensis Cei, 1950. The anurans were captured in the State of Mato Grosso, Brazil. Blood samples were obtained by cardiac puncture, and blood smears were examined for the presence of hemoparasites. The Trypanosoma rotatorium-like species in this study refers to a short-bodied trypomastigote that has a conspicuous undulating membrane but lacks a free flagellum; T. chattoni refers to a monomorphic parasite that has a rounded body, a kinetoplast adjacent to the nucleus, and a short flagellum.

Phylogeny of snake trypanosomes inferred by SSU rDNA sequences, their possible transmission by phlebotomines, and taxonomic appraisal by molecular, cross-infection and morphological analysis.

Blood examination by microhaematocrit and haemoculture of 459 snakes belonging to 37 species revealed 2.4% trypanosome prevalence in species of Viperidae (Crotalus durissus and Bothrops jararaca) and Colubridae (Pseudoboa nigra). Trypanosome cultures from C. durissus and P. nigra were behaviourally and morphologically indistinguishable. In addition, the growth and morphological features of a trypanosome from the sand fly Viannamyia tuberculata were similar to those of snake isolates. Cross-infection experiments revealed a lack of host restriction, as snakes of 3 species were infected with the trypanosome from C. durissus. Phylogeny based on ribosomal sequences revealed that snake trypanosomes clustered together with the sand fly trypanosome, forming a new phylogenetic lineage within Trypanosoma closest to a clade of lizard trypanosomes transmitted by sand flies. The clade of trypanosomes from snakes and lizards suggests an association between the evolutionary histories of these trypanosomes and their squamate hosts. Moreover, data strongly indicated that these trypanosomes are transmitted by sand flies. The flaws of the current taxonomy of snake trypanosomes are discussed, and the need for molecular parameters to be adopted is emphasized. To our knowledge, this is the first molecular phylogenetic study of snake trypanosomes.

Penetration of the salivary glands of Rhodnius domesticus Neiva & Pinto, 1923 (Hemiptera: Reduviidae) by Trypanosoma rangeli Tejera, 1920 (Protozoa: Kinetoplastida).

Penetration of the heteroxenous protozoan Trypanosoma rangeli into the salivary glands of its invertebrate host Rhodnius domesticus has been investigated here using different approaches. Electron microscopy showed that epimastigotes coming from the insect hemocoel cross the basal lamina that surrounds the salivary glands and penetrate through the gland cells cytoplasm. After reaching the gland lumen, epimastigote forms remain adhered to the gland cell microvilli by their flagella, while metacyclic trypomastigotes are found swimming free in the saliva. Analysis by flow cytometry, western blotting and hemolytic activity allowed to demonstrate the presence in T. rangeli of a hemolytic molecule with antigenic cross-reactivity with murine perforin, which could be used by the parasites to reach the salivary gland lumen. This molecule, which we named as rangelysin, has 120 kDa molecular weight, is able to induce hemolysis only in acidic pH, and is produced by both trypomastigote and epimastigote forms.

Trypanosoma rangeli: ultrastructure and activity of the mitochondrion.

Axenic cultures of pure Trypanosoma rangeli were used to investigate the relation between ultrastructure and activity in the mitochondrion. Every other day, ultrathin sections were obtained from cultivated flagellates and, subsequently, observed in order to register changes in the cytoarchitecture of the organelle. Culture samples were incubated in tetrazolium salts to determine the mitochondrion's metabolic state. The results show a high correspondence between mitochondrion ultrastructural shape and function of the same organelle in populations of T. rangeli maintained under in vitro conditions.

Further morphological studies on the behavior of Trypanosoma rangeli in the hemocytes of Rhodnius prolixus.

The hemocytes of Rhodnius prolixus were analyzed during the course of infection with the protozoan Trypanosoma rangeli. The following cell types were identified: prohemocyte, plasmatocyte, adipocyte, granular cell and oenocytoid. The number of these cells changes during the infection course thus indicating a cell response to infection of R. prolixus by T. rangeli. Transmission electron microscopy showed that plasmatocytes were able to ingest epimastigote forms of the parasite, which were then found within a parasitophorous vacuole. Amorphous material was seen within the vacuole suggesting that fusion of host cell lysosomes with the vacuole took place. Intravacuolar parasites in process of digestion were observed. In addition, reaction product indicative of the presence of acid phosphatase was observed in parasite-containing vacuoles. No dividing parasites were seen within the vacuole in contrast to what was observed outside the host cells.

Brazilian isolates of Trypanosoma (Megatrypanum) theileri: diagnosis and differentiation of isolates from cattle and water buffalo based on biological characteristics and randomly amplified DNA sequences.

We detected and cultivated isolates of Trypanosoma (Megatrypanum) theileri from cattle and water buffaloes in São Paulo state, southeastern Brazil, which were characterized by comparing morphological, growth and molecular features. Although isolates from cattle and water buffalo were morphologically indistinguishable, they differed in their growth characteristics. A dendrogram based on randomly amplified polymorphic DNA (RAPD) patterns indicated close-genetic relationships among all isolates from both species, which were all tightly clustered together and distant from Megatrypanum species from wild mammals. In addition, isolates within the T. theileri-cluster were clearly segregated into two host-associated groups. The sequence of a synapomorphic RAPD-derived DNA fragment (Tth625), which was shared by all T. theileri trypanosomes from cattle and buffalo but not detected in any of 13 other trypanosome species, was used as target for a conventional T. theileri-specific PCR assay. We also defined RAPD fragments (Tthc606 and Tthb606) that distinguished cattle from buffalo isolates. Thus, distinct growth features and genetic variability distinguished between isolates from cattle and water buffaloes of the same geographic origin, suggesting an association of these isolates with their host species. The trypanosomes from water buffalo reported here are the first T. theileri-like isolates from the Asiatic buffalo (Bubalus bubalis) to be continuously cultured and compared with cattle isolates using biological and molecular methods.

Physiological and morphological evidences for the presence acidocalcisomes in Trypanosoma evansi: single cell fluorescence and 31P NMR studies.

A new Ca(2+) intracellular store, the acidocalcisome, has been reported in trypanosomatids. It has been characterized physiologically as a Ca(2+) store sensitive to nigericin. The Ca(2+)/H(+)-ATPase is the system responsible for Ca(2+) accumulation, which depends on a pH gradient formed by ATP- and PPi-dependent proton pumps. In this work we present physiological and morphological evidences for the presence of acidocalcisomes in Trypanosoma evansi. The parasites were purified and loaded with the fluorescent dye Fura 2-AM in order to detect the intracellular changes of Ca(2+) levels in individual cells. The simultaneous incubation of T. evansi cells with ionomycin and nigericin led to large release of Ca(2+) (ca. 200 nM) from intracellular stores, which was not observed with either agent alone. On the other hand, no enhancement of the nigericin-induced Ca(2+) release was observed in the presence of oligomycin. Additionally, the pretreatment with bafilomycin decreases the nigericin-induced Ca(2+) release. These results confirm the presence of an intracellular non-mitochondrial acidic Ca(2+) storage compartment. These results suggest that H(+)-ATPase is involved in the process of Ca(2+) accumulation into the acidocalcisomes. Furthermore, the cells loaded with acridine orange exhibited abundant fluorescent vacuoles, which were sensitive to nigericin or bafilomycin A(1). Electronic transmission microscopy observations demonstrated the presence of electron dense particles in the parasites. High levels of inorganic pyrophosphate and triphosphate were detected in perchloric acid extracts of T. evansi by high resolution 31P NMR. Taken together, these results present the first evidence for the presence of acidocalcisomes in T. evansi.

An electron microscopic study of penetration by Trypanosoma rangeli into midgut cells of Rhodnius prolixus.

The process of interaction of the Choachi strain of Trypanosoma rangeli with intestinal epithelial cells of Rhodnius prolixus was analyzed in experiments carried out in vitro and in vivo. For the in vitro experiments small fragments of the anterior region of the posterior midgut were incubated in the presence of the parasites, fixed, and processed for observation with the scanning electron microscope. Parasites attached to the surface of some epithelial cells, especially to the extracellular membrane layers (perimicrovillar membranes), were observed. For the in vivo experiments insects were infected with cultures of T. rangeli, sacrificed at different time intervals, and then processed for scanning and transmission electron microscopy. An intimate contact between the parasites and the membrane layers was observed. The parasites penetrated into cells that showed an electronlucent cytoplasm and a damaged surface, moved within the cytoplasm of the epithelial cell, reached the basal region, crossed the basal lamina, and entered the hemocoel.

Molecular and morphological studies of Brazilian Trypanosoma evansi stocks: the total absence of kDNA in trypanosomes from both laboratory stocks and naturally infected domestic and wild mammals.

The kinetoplast DNA (kDNA) minicircle molecules of 14 Brazilian stocks of Trypanosoma evansi were studied by morphological approaches (Giemsa and 4'-6'-diamidino-2-phenylindole staining and transmission electron microscopy) and molecular approaches (probing with an oligonucleotide complementary to the minicircle origin of replication and polymerase chain reaction amplification of a minicircle sequence). All methods indicated the absence of both a typical kinetoplast and kDNA minicircles, even in a very small number of parasites of a single stock or in small numbers of copies of molecules per cell. We did not detect any altered kDNA molecules. There were no kDNA molecules in either old or new stocks of T. evansi maintained by successive passages in mice. Similarly, no kDNA minicircles were detected in trypanosomes in blood smears from naturally infected domestic and wild animals. Thus, the total absence of kDNA in Brazilian stocks of T. evansi from both domestic and wild mammals is probably the natural state of Brazilian T. evansi.

Ultrastructural alterations in the adrenal gland cortex of mice experimentally infected with a Venezuelan isolate of Trypanosoma evansi.

The ultrastructural study of adrenal gland from mice experimentally infected with Trypanosoma evansi, in addition to intravascular and intracellular trypanosomes, showed different degrees of cortical cell alterations and capillary wall modifications. Beside its biological scope, these results suggest a role for the adrenal cortex to partake in Surra's etiopathogenesis and describe for the very first time a T. evansi intracellular stage.