High Local Diversity of <i>Trypanosoma</i> in a Common Bat Species, and Implications for the Biogeography and Taxonomy of the <i>T. cruzi</i> Clade

<div><p>The <i>Trypanosoma cruzi</i> clade is a group of parasites that comprises <i>T. cruzi</i> sensu lato and its closest relatives. Although several species have been confirmed phylogenetically to belong to this clade, it is uncertain how many more species can be expected to belong into this group. Here, we present the results of a survey of trypanosome parasites of the bat <i>Artibeus jamaicensis</i> from the Panamá Canal Zone, an important seed disperser. Using a genealogical species delimitation approach, the Poisson tree processes (PTP), we tentatively identified five species of trypanosomes – all belonging to the <i>T. cruzi</i> clade. A small monophyletic group of three putative <i>Trypanosoma</i> species places at the base of the clade phylogeny, providing evidence for at least five independent colonization events of these parasites into the New World. <i>Artibeus jamaicensis</i> presents a high diversity of these blood parasites and is the vertebrate with the highest number of putative trypanosome species reported from a single locality. Our results emphasize the need for continued efforts to survey mammalian trypanosomes.</p></div

Phylogeny of the <i>Trypanosoma cruzi</i> clade.

<p>Maximum likelihood tree constructed with concatenated 18S rRNA and gGAPDH genes. Numbers on the branches represent support values corresponding to bootstrap replicates (right) and Bayesian posterior probabilities (left). Clades highlighted with colored boxes correspond to trypanosomes detected in Panamá. Collapsed branches at the base of the phylogeny indicate low support for the placement of the three Panamian lineages. The symbol//on the branch to the outgroup indicates a shortened branch. GenBank accession numbers of the samples used in this phylogeny are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108603#pone.0108603.s001" target="_blank">Table S1</a>.</p

Putative species delimitation of members of the <i>Trypanosoma cruzi</i> clade.

<p>Maximum likelihood phylogeny with Bayesian support values presented for all 18 lineages recognized as species for the PTP analyses. Monophyletic groups in red indicate a single putative species as well as terminal branches in blue. Names of terminals indicate codes of the samples. Names in bold after a | symbol are taxonomic or geographic identifiers of the putative species. Arrows indicate the 5 putative species found in <i>Artibeus jamaicensis</i> in Panamá.</p

Five independent colonization events of <i>Trypanosoma cruzi</i> clade members to the New World.

<p>Cladogram of species belonging to the <i>Trypanosoma cruzi</i> clade, depicting in red are the branches that indicate dispersal events to the Americas with grey branches denoting the species that are not present in the New World. Note that <i>T. dionisii</i> 2 and <i>T. conorhini</i> are distributed in the New and Old Worlds <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108603#pone.0108603-Hamilton3" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108603#pone.0108603-Hamilton4" target="_blank">[43]</a>. Branch lengths in the cladogram are not proportional to divergence; for branch length estimates refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108603#pone-0108603-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108603#pone-0108603-g002" target="_blank">2</a>.</p

Immunofluorescence staining for markers of epithelial origin of Tb 1 Lu and airway epithelial cells from <i>C. perspicillata</i> (CarperAEC.B) and <i>E. helvum</i> (EidheAEC.B) prior to subcloning.

<p>The markers used to confirm epithelial origin were cytokeratin (CK, red) and zonula occludens-1 (ZO-1, green); nuclei are counterstained with DAPI (blue). Expression of both markers is present in all cell lines generated by the described methods, indicating an epithelial origin. By contrast, the commercially available Tb 1 Lu does not show expression of the respective markers.</p

Virus infection studies with vesicular stomatitis virus (VSV) (A, B) and Rift Valley fever virus (RVFV) (C, D), and at MOI of 0.1 (left side) and 0.001 (right side), in subclones of immortalized <i>C. perspicillata</i> airway epithelial cells (subclone 3, designated CarperAEC.B-3) and <i>E. helvum</i> airway epithelial cells (subclone 1, designated EidheAEC.B-1).

<p>Viruses were detected by quantitative real-time RT-PCR. All cells were infectable and able to support replication of RVFV and VSV.</p

Bat Airway Epithelial Cells: A Novel Tool for the Study of Zoonotic Viruses

<div><p>Bats have been increasingly recognized as reservoir of important zoonotic viruses. However, until now many attempts to isolate bat-borne viruses in cell culture have been unsuccessful. Further, experimental studies on reservoir host species have been limited by the difficulty of rearing these species. The epithelium of the respiratory tract plays a central role during airborne transmission, as it is the first tissue encountered by viral particles. Although several cell lines from bats were established recently, no well-characterized, selectively cultured airway epithelial cells were available so far. Here, primary cells and immortalized cell lines from bats of the two important suborders Yangochiroptera and Yinpterochiroptera, <i>Carollia perspicillata</i> (Seba's short-tailed bat) and <i>Eidolon helvum</i> (Straw-colored fruit bat), were successfully cultured under standardized conditions from both fresh and frozen organ specimens by cell outgrowth of organ explants and by the use of serum-free primary cell culture medium. Cells were immortalized to generate permanent cell lines. Cells were characterized for their epithelial properties such as expression of cytokeratin and tight junctions proteins and permissiveness for viral infection with Rift-Valley fever virus and vesicular stomatitis virus Indiana. These cells can serve as suitable models for the study of bat-borne viruses and complement cell culture models for virus infection in human airway epithelial cells.</p></div

Species for which airway epithelial cells were established and their geographic distribution.

<p>A. Seba's short-tailed bat (<i>C. perspicillata</i>), and B. Straw-colored fruit bat (<i>E. helvum</i>) (upper row), and their distribution (lower row) (map adapted from IUCN Red List of Threatened Species, <a href="http://www.iucnredlist.org" target="_blank">http://www.iucnredlist.org</a>).</p

Establishment of airway epithelial cell culture by outgrowth from trachea specimens.

<p><b>A</b> Trachea tissue sample in cell culture dish viewed from above (left side) with outgrowth of primary airway epithelial cells from the mucosal layer (right side, 100× magnification) (here: <i>E. helvum</i>) <b>B</b> Immortalized and subcloned airway epithelial cells from <i>E. helvum</i>, subclone 1 (designated EidheAEC.B-1) <b>C</b> Immortalized and subcloned airway epithelial cells from <i>C. perspicillata</i>, subclone 3 (designated CarperAEC.B-3). Black bars represent 50 µm.</p

Characteristics of bat species chosen for the establishment of bat airway epithelial cells.

<p>Characteristics of bat species chosen for the establishment of bat airway epithelial cells.</p