Genus Endotrypanum Research Articles

Porcisia transmission by prediuresis of sand flies.

Parasites of the genus Porcisia, together with the genus Endotrypanum, form a sister clade to the species-rich and medically important genus Leishmania. Both Porcisia species, P. hertigi and P. deanei, are dixenous parasites of Neotropical porcupines. Almost 50 years after their first discovery, knowledge of their life cycle remains poor and their insect vectors are unknown. Because competent vectors of their closest phylogenetic relatives, genera Endotrypanum and Leishmania, are phlebotomine sand flies (Diptera: Psychodidae) and/or biting midges (Diptera: Ceratopogonidae), we examined here the potential of both sand flies and biting midges to transmit Porcisia parasites. The insects (Lutzomyia longipalpis, L. migonei and Culicoides sonorensis) were exposed to parasites through the chicken skin membrane and dissected at various time intervals post bloodmeal. Potentially infected females were also allowed to feed on the ears of anaesthetized BALB/c mice and the presence of parasite DNA was subsequently confirmed in the mice by PCR. Porcisia hertigi did not survive defecation in L. longipalpis or L. migonei, suggesting that these sand fly species are unlikely to serve as natural vectors of this parasite. Similarly, P. hertigi infections were lost in Culicoides midges. In contrast, mature P. deanei infections developed in 51-61% of L. longipalpis females, 7.3% of L. migonei females and 7.7% of Culicoides sonorensis females. In all three vector species, P. deanei colonized predominantly Malpighian tubules and produced metacyclic infective forms. Transmission of P. daenei to BALB/c mice was demonstrated via the prediuresis of L. longipalpis females. This mode of transmission, as well the colonization of Malpighian tubules as the dominant tissue of the vector, is unique among trypanosomatids. In conclusion, we demonstrated the vector competence of L. longipalpis for P. deanei but not for P. hertigi, and further studies are needed to evaluate competence of other Neotropical vectors for these neglected parasites.

Comparative genomics of Leishmania (Mundinia)

BackgroundTrypanosomatids of the genus Leishmania are parasites of mammals or reptiles transmitted by bloodsucking dipterans. Many species of these flagellates cause important human diseases with clinical symptoms ranging from skin sores to life-threatening damage of visceral organs. The genus Leishmania contains four subgenera: Leishmania, Sauroleishmania, Viannia, and Mundinia. The last subgenus has been established recently and remains understudied, although Mundinia contains human-infecting species. In addition, it is interesting from the evolutionary viewpoint, representing the earliest branch within the genus and possibly with a different type of vector. Here we analyzed the genomes of L. (M.) martiniquensis, L. (M.) enriettii and L. (M.) macropodum to better understand the biology and evolution of these parasites.ResultsAll three genomes analyzed were approximately of the same size (~ 30 Mb) and similar to that of L. (Sauroleishmania) tarentolae, but smaller than those of the members of subgenera Leishmania and Viannia, or the genus Endotrypanum (~ 32 Mb). This difference was explained by domination of gene losses over gains and contractions over expansions at the Mundinia node, although only a few of these genes could be identified. The analysis predicts significant changes in the Mundinia cell surface architecture, with the most important ones relating to losses of LPG-modifying side chain galactosyltransferases and arabinosyltransferases, as well as β-amastins. Among other important changes were gene family contractions for the oxygen-sensing adenylate cyclases and FYVE zinc finger-containing proteins.ConclusionsWe suggest that adaptation of Mundinia to different vectors and hosts has led to alternative host-parasite relationships and, thereby, made some proteins redundant. Thus, the evolution of genomes in the genus Leishmania and, in particular, in the subgenus Mundinia was mainly shaped by host (or vector) switches.

Vetores de tripanosomatídeos (Kinetoplastida: Trypanosomatidae) em Tabatinga, Amazonas, Brasil

Flebotomíneos transmissores de tripanosomatídeos foram capturados no município de Tabatinga, com armadilhas luminosas do tipo CDC e em coletas manuais por aspiração em bases das árvores. Em agosto de 2010 e 2011, foram capturados 4.542 flebotomíneos (♂: 71,9% e ♀: 28,1%) vetores ou suspeitos de transmitirem tripanosomatídeos, pertencentes a 13 espécies. A espécie mais abundante foi Lutzomyia umbratilis com 78,7%, seguida de L. dendrophyla com 10,5%, L. davisi 3,4%, L. shannoni 2,2%, L. yuilli 1,6%. Um total de 576 (74%) fêmeas do gênero Lutzomyia foram dissecadas, sendo observada infecção natural por tripanosomatídeos em 44 indivíduos. Observou-se no trato digestório dos flebotomíneos a presença de sangue em decomposição em 86,3%; sendo que no que se refere aos ovários, 59% mostravam-se em desenvolvimento. Análise isoenzimática de dez isolados, demonstrou perfis similares a Leishmania colombiensis e Endotrypanum sp., no entanto, o perfil do locus MDH demonstrou maior similaridade para o gênero Endotrypanum. Esta é a primeira investigação da fauna entomologia e tentativa de isolamento de tripanosomatídeos no município de Tabatinga, área de tríplice fronteira com o Brasil.

An appraisal of the taxonomy and nomenclature of trypanosomatids presently classified as Leishmania and Endotrypanum.

We propose a taxonomic revision of the dixenous trypanosomatids currently classified as Endotrypanum and Leishmania, including parasites that do not fall within the subgenera L. (Leishmania) and L. (Viannia) related to human leishmaniasis or L. (Sauroleishmania) formed by leishmanias of lizards: L. colombiensis, L. equatorensis, L. herreri, L. hertigi, L. deanei, L. enriettii and L. martiniquensis. The comparison of these species with newly characterized isolates from sloths, porcupines and phlebotomines from central and South America unveiled new genera and subgenera supported by past (RNA PolII gene) and present (V7V8 SSU rRNA, Hsp70 and gGAPDH) phylogenetic analyses of the organisms. The genus Endotrypanum is restricted to Central and South America, comprising isolates from sloths and transmitted by phlebotomines that sporadically infect humans. This genus is the closest to the new genus Porcisia proposed to accommodate the Neotropical porcupine parasites originally described as L. hertigi and L. deanei. A new subgenus Leishmania (Mundinia) is created for the L. enriettii complex that includes L. martiniquensis. The new genus Zelonia harbours trypanosomatids from Neotropical hemipterans placed at the edge of the Leishmania-Endotrypanum-Porcisia clade. Finally, attention is drawn to the status of L. siamensis and L. australiensis as nomem nudums.

Phylogenetic relationships of Leishmania species based on trypanosomatid barcode (SSU rDNA) and gGAPDH genes: Taxonomic revision of Leishmania (L.) infantum chagasi in South America

Phylogenetic studies on trypanosomatid barcode using V7V8 SSU rRNA and gGAPDH gene sequences have provided support for redefining some trypanosomatid species and positioning new isolates. The genus Leishmania is a slow evolving monophyletic group and including important human pathogens. The phylogenetic relationships of this genus have been determined by the natural history of its vertebrate hosts, vector specificity, clinical manifestations, geographical distribution and molecular approaches using different markers. Thus, in an attempt to better understand the phylogenetic relationships of Leishmania species, we performed phylogenetic analysis on trypanosomatid barcode using V7V8 SSU rRNA and gGAPDH gene sequences among a large number of Leishmania species and also several Brazilian visceral Leishmania infantum chagasi isolates obtained from dogs and humans. Our phylogenetic analysis strongly suggested that Leishmania hertigi and Leishmania equatoriensis should be taxonomically revised so as to include them in the genus Endotrypanum; and supported ancient divergence of Leishmania enriettii. This, together with recent data in the literature, throws light on the discussion about the evolutionary southern supercontinent hypothesis for the origin of Leishmania ssp. and validates L. infantum chagasi from Brazil, thus clearly differentiating it from L. infantum, for the first time.

Phylogenic analysis of the genus Leishmania by cytochrome b gene sequencing

In a previous report ( Luyo-Acero et al., 2004), we demonstrated that cytochrome b (Cyt b) gene analysis is an effective method for classifying several isolates of the genus Leishmania; hence, we have further applied this method to other Leishmania species in an effort to enhance the accuracy of the procedure and to construct a new phylogenic tree. In this study, a total of 30 Leishmania and Endotrypanum WHO reference strains, clinical isolates from our patients assigned to 28 strains (human and non-human pathogenic species) and two species of the genus Endotrypanum were analyzed. The Cyt b gene in each sample was amplified by PCR, and was then sequenced by several primers, as reported previously. The phylogenic tree was constructed based on the results obtained by the computer software MEGA v3.1 and PAUP* v4.0 Beta. The present phylogenic tree was almost identical to the traditional method of classification proposed by Lainson and Shaw (1987). However, it produces the following suggestions: (1) exclusion of L. ( Leishmania) major from the L. ( L.) tropica complex; (2) placement of L. tarentolae in the genus Sauroleishmania; (3) L. ( L.) hertigi complex and L. ( V.) equatorensis close to the genus Endotrypanum; (4) L. ( L.) enrietti, defined as L. ( L.) mexicana complex, placed in another position; and (5) L. ( L.) turanica and L. ( L.) arabica are located in an area far from human pathogenic Leishmania strains. Cyt b gene analysis is thus applicable to the analyzing phylogeny of the genus Leishmania and may be useful for separating non-human pathogenic species from human pathogenic species.

Identification of endotrypanum species from a sloth, a squirrel and Lutzomyia sandflies in ecuador by PCR amplification and sequencing of the mini-exon gene.

PCR amplification and nucleotide sequencing of the mini-exon gene revealed that four strains isolated from a sloth (Choloepus hoffmanni), a squirrel (Sciurus granatensis) and two sandflies (Lutzomyia hartmanni) in Ecuador were indistinguishable from Endotrypanum monterogeii. Another strain isolated from Lu. hartmanni showed the high sequence similarity to E. schaudinni. Since three of these strains have been previously identified as Leishmania (Viannia) equatorensis, the results demonstrate that L. (V.) equatorensis is genetically closely related to the genus Endotrypanum. The present study also indicates that Endotrypanum species are distributed in arboreal animals and sandflies in Ecuador, and that mini-exon gene amplification is useful for epidemiological studies of Leishmania and Endotrypanum in the New World.

The Leishmania hertigi (Kinetoplastida; Trypanosomatidae) complex and the lizard Leishmania: their classification and evidence for a neotropical origin of the Leishmania-Endotrypanum clade.

The relationships of the Leishmania hertigi complex and the lizard Leishmania species to the main groups of mammalian Leishmania and Endotrypanum parasites were examined. Restriction fragment length polymorphisms and sequences of small subunit ribosomal RNA genes and hybridization studies of kinetoplast DNA indicated that the L. hertigi complex was more closely related to the genus Endotrypanum than to the genus Leishmania. The lizard Leishmania species were found to be at the crown of the Leishmania tree. The data provides strong evidence for a Neotropical origin of the Endotrypanum/Leishmania clade since the parasites closest to the root of the tree are all found exclusively in the Neotropics. The evolution of the Leishmania/Endotrypanum clade in relation to the evolution of the known hosts of these parasites is discussed.

Enzyme polymorphism in Endotrypanum and numerical analysis of isoenzyme data.

In this study, we have analysed enzyme polymorphism among a group of protozoan parasites of the genus Endotrypanum (Kinetoplastida: Trypanosomatidae). Seventeen stocks of Endotrypanum spp. isolated from sloths (Choloepus didactylus and C. juruanus) in the Amazon Region of Brazil were analysed by enzyme electrophoresis, and their electromorphic profiles were compared with reference strains reported previously. The 16 enzymic loci were analysed, and the strains were classified into zymodemes, each representing parasites with unique enzyme profiles. Each zymodeme was considered as an elementary taxon, and using numerical analyses (cladistic, agglomerative hierarchical and ordination techniques) the genus was shown to be monophyletic and the 12 zymodemes characterized could be divided into 3 groups (A, B, C). The heterogeneous population (which may represent a complex of parasite species or strains variants) showed, however, no correlation with the origin (i.e. host species involved or geographic area of isolation) of Endotrypanum stocks. Eight isolates of Endotrypanum sp. from Rondônia State (Brazil) and a parasite strain from Panama were clustered together into a zymodeme, which was phenetically closely related to the E. monterogeii from Costa Rica. The data indicate that E. schaudinni is a species complex.

Ultrastructural and stereological analysis of trypanosomatidis of the genus Endotrypanum

Culture forms of four strains of Endotrypanum (E. schaudinni and E. monterogeii) were processed for transmission electron microscopy and analyzed at the ultrastructural level. Quantitative data about some cytoplasmic organelles were obeined by stereology. All culture forms were promastigotes. In their cytoplasm four different organelles could be found: lipid inclusions (0,2-0,4 µm in diameter), mebrane-bounded vacuoles (0.10-0,28 µm in diameter), glycosomes (0,2-0,3 µm in diameter), and the mitochondrion. The kenetoplast appears as a thin band, except for the strain IM201, which possesses a broader structure, and possibly is not a member of this genus. Clusters of virus-like particles were seen in the cytoplasm of the strain LV88. The data obtained show that all strains have the typical morphological feature of the trypanosomatids. Only strain IM201 could be differentiated from the others, due to its larger kenetoplast-DNA network and its large mitochondrial and glycosomal relative volume. The morphometrical data did not allow the differentiation between E. schaudinni (strains IM217 and M6226) and E. monterogeii (strain LV88).

Evolution of nuclear DNA and the occurrence of sequences related to new small chromosomal DNAs in the trypanosomatid genus Endotrypanum

Comparisons of nuclear DNA restriction fragment patterns were used to examine the evolutionary relatedness among 17 strains previously identified as Endotrypanum, a trypanosomatid parasite of sloths. Fragments were obtained with 6 restriction enzymes and analyzed by Southern blotting with hybridization probes from three loci. An estimate of the percent nucleotide sequence divergence among strains, Δ, was calculated and used to construct molecular evolutionary trees. The 17 isolates fell into four distinct groups, one of which (group D) showed no more relationship to groups A–C than it did to other genera ( Leishmania, Crithidia, Leptomonas, Trypanosoma), being too distant to be resolved with this method. These and other data suggest that group D may not actually be Endotrypanum. Molecular karyotype analysis revealed considerable variation among the chromosomes of these strains. One strain (LV88, group B) contained a linear 70-kb chromosome not evident in other isolates. Hybridization probes specific for this chromosome (LV88-70) were developed and revealed that related sequences were present at high levels in group B isolates and low levels in group A isolates, although a complex hybridization pattern was evident. Sequences related to LV88-70 were not present in groups C and D, nor in Leishmania major, showing that this DNA has a disjunct distribution which curiously parallels that of virus-like particles present in these isolates.

Monoclonal Antibodies Specific for Members of the Genus Endotrypanum

Twenty-six monoclonal antibodies were produced against membrane-enriched preparations of Endotrypanum schaudinni or Endotrypanum sp. promastigotes. Fifteen of these monoclonal antibodies (E1-E15) reacted only with the standard strain of E. schaudinni, M6159. Monoclonal antibodies E16-E26 were considered Endotrypanum specific; no cross reactivity was detected with any other genus of the family Trypanosomatidae (Leishmania, Trypanosoma, Leptomonas, Herpetomonas or Crithidia) by dot-blot radioimmune assay. By indirect immunofluorescence assay, the antigens recognized by Endotrypanum specific monoclonal antibodies appear to be associated with the surface of the parasite. Based on Western blot analysis, 4 antigenic molecules ranging in molecular weight from 24 kD to 160 kD were identified by monoclonal antibodies specific for the strain of E. schaudinni, M6159. Monoclonal antibodies specific for the genus Endotrypanum identified an antigen of molecular weight 48 kD as well as a diffuse component migrating with an apparent molecular weight of 64-200 kD.

Contribución al estudio de Endotrypanum schaudinni (Trypanosomidae)

A chronological review of the existing literature on Endotrypanum schaudinni MESNIL and BRIMONT 1908 is made. An arrangement of the existing knowledge on these interesting trypanosomes is attempted and further facts are contributed. The taxonomic position and characteristics of their vertebrate hosts, Bradypus and Choloepus, is given as well as some observations on the biology and geographical distribution on the specimens captured this country. The morphology of the parasite has been observed both fresh as well as stained preparations, and trypanosome forms have been reported Choloepus hoffmanni for the first time. Intracellular forms are reported only those slides which were prepared immediately after obtaining the blood. The technique and the different culture media employed the primary isolation of the hemoparasite are given. The harmful effects of anti-clotting agents the culture media is noticed. A study of the culture forms is carried out both on fresh as well as on stained preparations, reporting our strains only the leshmania and leptomona forms. A comment is made relation to the trypanosome forms that had been reported by CUNHA and MUNIZ (4). Different laboratory animals were inoculated with blood of infected Choloepus and with culture forms without being able to demonstrate infection any of them, probably due to their great specificity toward edentates inferred easily from their strict intracellular parasitism. In addition, comments are made on the importance of the physiological state of the animals to be inoculated. An adult Choloepus and a human being were inoculated with fresh culture forms but negative results were obtained. This might be due to a 1055 of virulence of the in vitro strain or to a state of premunition of the adult specimen to a protozoosis. Recently isolated culture forms were inoculated into the vitellin sac of the chick embryo with negative results. With respect to its evolutive cycle, the parasite does not present division forms the peripheral blood stream nor were its tissular forms found the histological examination. The finding of parasite nests a breast of Bradypus griseus griseus does not make its evolution the vertebrate host any clearer to us due to the fact that the sloth was parasitized with another trypanosome and that the presence of a kinetoplast could not be demonstrated these forms, all of which suggests the possibility of a toxoplasmosis. In exalated blood kept at room temperature the parasites leave the erythrocytes and show a better locomotive apparatus. This we relate to its evo­lutive cycle since this phenomenon probaly takes place the intestinal tract of some intermediary arthropod host. Several ectoparasites were collected from Choloepus, among them a tick of the genus Amblyomma. In Bradypus, several arthropods that live intimate relationship with this vertebrate are reported, such as Cryploses choloepi, among them a tick of the genus Amblyomma. In Bradypus, several arthropods that live intimate relationship with this vertebrate are reported, such as Cryptoses choloepi, Trichillum bradyporum, and a coleopterus of the family Scarabeidae. In addition, Amblyomma gertschi was found and was carefully studied since it was the only hematophagous ectoparasite. An unsuccesful effort is made at finding a hemipterus, Belminus costa­rincensis, that had been reported a sloth. Although the search for the vector of this parasitosis was fruitless, we are convinced of its existence and will continue searching for it. We carried out a xenodiagnosis on Choloepus, with Triatoma infestans and Triatoma dimidiata, without being able to obtain the evolution of the parasite. The main characteristics of the genus Endotrypanum are given but it cannot be definitely placed the family Trypanosomidae since its evolutive cycle is not known.