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

Abstract

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.

Figure 1

Light microscopy of Trypanosoma terrestris. Photomicrographs (Giemsa-stained) of LIT culture forms from Trypanosoma terrestris sp. nov. Epimastigote forms from logarithmic phase arranged in rosettes with kinetoplast and nucleus shown (a-d) without free flagellum (c-d) and flagellar sheath (*). Metacyclic trypomastigote forms from stationary phase (e-g). N, nucleus; K, kinetoplast; F, flagellum.

Figure 2

Scanning electron microscopy on T. terrestris . Epimastigote forms in rosettes (a-f) or separate (f-h), twisted epimastigote (g) in metacyclogenesis (i, j, m) and flagellar sheath (h). Cytosome indicated by arrows (h, m). Metacyclic trypomastigote forms with long free flagellum (j, n, o) and undeveloped undulating membrane, indicated by arrows (n, o). Scale bar 10 μm.

Figure 3

Transmission electron microscopy on T. terrestris . Logarithmic and stationary phase from epimastigote forms (a, b, d, f) and trypomastigote forms (c, e). Ultrastructural organization, mitochondria, nucleus and kinetoplast common to Trypanosoma (a-d). Large and loose kinetoplast (c, d). Multiple lysosomes (b, d), reservosomes (b), cytosome (b). Flagellum and paraxonemal structure (c-e), well developed flagellar pocket (d-e) and basal bodies in a structure that shelters the flagellum in epimastigote forms (f). Contractile vacuole (*). M, mitochondria; N, nucleus; K, kinetoplast; F, flagellum; R, reservosome; L, lysosome; Cy, cytosome; FP, flagellar pocket; PR, paraxial rod; BB, basal bodies.

Figure 4

Phylogenetic tree of a new trypanosome species, Trypanosoma terrestris , from Lowland tapir. Phylogenetic tree based on concatenated SSU rDNA and gGAPDH gene sequences of 51 trypanosome isolates using non-trypanosome trypanosomatids as outgroup (2898 characters and 875 parsimony-informative sites), which was used in parsimony and Bayesian methods. Numbers at nodes are support values for the major branches (bootstrap/posterior probability; 500 replicates). T. terrestris clades are bold and in gray box.