Figures
Abstract
Background
Bats are a highly successful, globally dispersed order of mammals that occupy a wide array of ecological niches. They are also intensely parasitized and implicated in multiple viral, bacterial and parasitic zoonoses. Trypanosomes are thought to be especially abundant and diverse in bats. In this study, we used 18S ribosomal RNA metabarcoding to probe bat trypanosome diversity in unprecedented detail.
Methodology/Principal Findings
Total DNA was extracted from the blood of 90 bat individuals (17 species) captured along Atlantic Forest fragments of Espírito Santo state, southeast Brazil. 18S ribosomal RNA was amplified by standard and/or nested PCR, then deep sequenced to recover and identify Operational Taxonomic Units (OTUs) for phylogenetic analysis. Blood samples from 34 bat individuals (13 species) tested positive for infection by 18S rRNA amplification. Amplicon sequences clustered to 14 OTUs, of which five were identified as Trypanosoma cruzi I, T. cruzi III/V, Trypanosoma cruzi marinkellei, Trypanosoma rangeli, and Trypanosoma dionisii, and seven were identified as novel genotypes monophyletic to basal T. cruzi clade types of the New World. Another OTU was identified as a trypanosome like those found in reptiles. Surprisingly, the remaining OTU was identified as Bodo saltans–closest non-parasitic relative of the trypanosomatid order. While three blood samples featured just one OTU (T. dionisii), all others resolved as mixed infections of up to eight OTUs.
Conclusions/Significance
This study demonstrates the utility of next-generation barcoding methods to screen parasite diversity in mammalian reservoir hosts. We exposed high rates of local bat parasitism by multiple trypanosome species, some known to cause fatal human disease, others non-pathogenic, novel or yet little understood. Our results highlight bats as a long-standing nexus among host-parasite interactions of multiple niches, sustained in part by opportunistic and incidental infections of consequence to evolutionary theory as much as to public health.
Author summary
Bats make up a mega-diverse, intensely parasitized order of volant mammals whose unique behavioural and physiological adaptations promote infection by a vast array of microorganisms. Trypanosomes stand out as ancient protozoan parasites of bats. As cryptic morphology, low parasitaemia and selective growth in culture have recurrently biased survey, we used 18S ribosomal RNA metabarcoding to resolve bat trypanosomatid diversity in Atlantic Forest fragments of southeast Brazil. Next to several unknown species, our deep sequence-based detection and assignment protocol recognized multiple known human-pathogenic trypanosomes, another linked to reptile hosts as well as a non-parasitic kinetoplastid in the blood of various phyllostomid bats. The striking permissivity exposed here, in a region where bat trypanosomes recently featured in a fatal case of Chagas disease, compels further research on bats’ role in the dispersal and spill-over of various microorganisms among humans and wildlife.
Citation: Dario MA, Moratelli R, Schwabl P, Jansen AM, Llewellyn MS (2017) Small subunit ribosomal metabarcoding reveals extraordinary trypanosomatid diversity in Brazilian bats. PLoS Negl Trop Dis 11(7): e0005790. https://doi.org/10.1371/journal.pntd.0005790
Editor: Carlos A. Buscaglia, Instituto de Investigaciones Biotecnológicas, ARGENTINA
Received: April 13, 2017; Accepted: July 10, 2017; Published: July 20, 2017
Copyright: © 2017 Dario et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All sequences have been deposited in the NCBI Sequence Read Archive (SRA) under accession numbers SRR5451077-SRR5451120.
Funding: This study was funded by the Oswaldo Cruz Foundation (Fiocruz), the National Council for Scientific and Technological Development (CNPq) and the Carlos Chagas Filho Research Support Foundation of the State of Rio de Janeiro (FAPERJ). A doctoral grant was provided by CNPq and a SWP grant was provided by FAPERJ to MAD. PS is supported by NIH project 1R15AI105749-01A1. AMJ is funded by FAPERJ (“Cientista do Nosso Estado”) and by CNPq (“Bolsista de Produtividade, Nível 1”). RM is supported by PAPES VI Fiocruz/CNPq project 407623/2012-4. The funders had no role in study design, data collection and analysis, decision to publish, or manuscript preparation.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Trypanosoma cruzi is the etiological agent of Chagas disease, a complex zoonosis that continues to take dozens of human lives each day [1]. Alongside its close relative Trypanosoma cruzi marinkellei in the Schizotrypanum subgenus, this important protozoan flagellate belongs to a broader, inter-continental group (the “T. cruzi clade”) of ancient endoparasites found to infect the mammalian fauna far and wide [2–3]. Infections have been reported in primates of Africa [4], marsupials of Australia [5] and a multitude of terrestrial mammals across the Americas [6], but most of this striking spread in host diversity tallies to few taxa within the clade (above all to T. cruzi sensu stricto, i.e., T. cruzi, and to T. rangeli).
The majority of T. cruzi clade diversity is found in bats. Chiroptera are known to carry both generalists such as T. cruzi and T. rangeli as well as multiple bat-restricted species—some abundant (e.g., T. c. marinkellei, T. dionisii and T. erneyi), others rare (e.g. T. livingstonei and T. wauwau) [3, 7–8]. Chiropteran immunity is unique with respect to other mammalian genera, coincident perhaps with physiological adaptations to flying [9]. Several features of bat immunity may predispose bats to long-term asymptomatic infections [10] with viruses [11–12], bacteria [13–14], fungi [15–16], protozoa [17–18] and helminths [19–20], several of which cause disease in humans and animals [21].
Given the diversity of bat-infecting T. cruzi-clade trypanosomes throughout the New and Old Worlds, many now accredit the Chiroptera with a fundamental role in the evolution of this parasite group [22]. In fact, the most parsimonious explanation to date for the origin and past expansion of the T. cruzi clade suggests a common ancestral lineage of bat-restricted trypanosomes that diversified into several independent lineages that on rare occasion switched into other terrestrial mammal hosts [17]. Bats’ recurrent interaction with other mammals and their various ectoparasites are thought to have afforded enough opportunity for at least five such switching or “seeding” events, likely since the early Eocene (54 to 48 million years ago) [7].
Many trypanosomes from bats are morphologically indistinguishable, often described simply as “T. cruzi-like” in the past [23]. As mixed species/genotype infections are probably common but overlooked or mistaken, molecular barcoding presents expedient recourse in resolving intricate trypanosomatid taxonomy and ecology. Metabarcoding couples classic molecular barcoding with next generation sequencing techniques [24–25] to generate thousands of sequence reads from a single sample [26–27]. These reads correspond to the diversity and abundance of organisms infecting the host individual [28–30].
In this study, we applied next-generation metabarcoding methods to the most bat-diverse (per area) biome of Brazil [31]. We focused on a degraded section of Atlantic Forest in Espírito Santo (ES) state where terrestrial mammals appear reduced in abundance as well as in T. cruzi infection. A fatal case of human T. cruzi (I-IV) and T. dionisii coinfection [32] immediately predated the bat trypanosome survey by 18S ribosomal RNA deep sequencing in this region.
Methods
Ethical statement
The sampling procedures reported herein were authorized by the Brazilian Institute of the Environment and Renewable Natural Resources (IBAMA) under license no. 19037–1 (23-05-2009). Euthanasia and blood collection met guidelines set by the Federal Council of Veterinary Medicine, Resolution 1000 (11-05-2012), in accordance to Federal Law 11.794/2008. All procedures followed protocols approved by the Oswaldo Cruz Foundation (Fiocruz) Ethics Committee for Animal Research (L0015-07).
Study area, bat capture and sampling
Bat captures were carried out in two periods of 2015: June (dry season) and November (rainy season). Mist nets were opened upon sunset for four hours on two consecutive nights at each study location. A total of 108 bats were captured using ten mist nets (3 x 9 m, 35 mm mesh) placed along forest edges near banana and coffee crops at three different rural locations in Guarapari municipality, ES state, southeast Brazil: Rio da Prata (350 m a.s.l.), where a fatal case of Chagas disease occurred in 2012; Buenos Aires (250 m a.s.l.), where reports of triatomine invasion have increased in recent years; and Amarelos (at sea level), where triatomines have not been reported from the domestic zone (based on records by the Zoonosis Control Center, Guarapari municipality, ES) (S1 Fig).
Taxonomic identification by morphology followed [33] and a maximum of ten individuals per species (per site) were kept for further sampling, as specified by law. Once anesthetized with acepromazine (2%) in 9:1 ketamine hydrochloride (10%), these individuals were cleared of fur in the pectoral region (by scalpel) and sterilized with antiseptic soap and iodinated ethanol (70%) for blood withdrawal by cardiac puncture. Within the safety area of a flame, 300 μl blood was collected into sterile 1.5 ml vials and stabilized in two parts (i.e., 600 μl) 6 M Guanidine-HCl, 0.2 M EDTA solution for storage at -20°C. All bats used in these analyses received a collection number with the initials of the collector (RM) and were prepared for fluid preservation. This material will be subsequently deposited at the mammal collection of Museu Nacional, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
18S rRNA amplification and deep sequencing
DNA was purified from 90 guanidine-EDTA blood lysates in DNeasy mini spin columns (Qiagen), with each of nine extraction rounds including one negative control. Purified DNA samples were then PCR-amplified with primers 5’-TGGGATAACAAAGGAGCA-3’ (forward) and 5’-CTGAGACTGTAACCTCAAAGC-3’ (reverse) for 30 cycles of 94°C (30 s), 55°C (60 s) and 72°C (90 s) to target a trypanosome-specific, ~556 bp region of the 18S rRNA gene as established in [5]. For a subset of samples, a wider, ~927 bp region (encompassing the ~556 bp above) was first targeted with external primers 5’-CAGAAACGAAACACGGGAG-3’ (forward) and 5’-CCTACTGGGCAGCTTGGA-3’ (reverse) at equivalent cycling conditions to form a nested (two-round) PCR amplification procedure following [34]. Sterile water (2x) and sample-free eluate from prior DNA purification (1x) were used to provide three negative controls per 20-sample PCR reaction. Amplicons were single-end barcoded [35], purified by agarose gel electrophoresis (PureLink Quick Gel Extraction Kit, Invitrogen), quantified by fluorometric assay (Qubit 2.0, Thermo Fisher Scientific) and pooled to equimolar concentration for multiplexed, paired-end (2 x 300 bp) sequencing on the Illumina MiSeq platform (Reagent Kit v2).
Species delimitation and phylogenetic analysis
Amplicon sequences were filtered to retain only full-length reads of ≥ 99.9% base call accuracy by windowed trimming in Sickle [36], verified for quality in FastQC [37] and mapped against a Trypanosoma spp. reference collection from SILVA v119 [38] using Bowtie 2 [39]. Operational Taxonomic Unit (OTU) construction proceeded by UPARSE algorithm in USEARCH [40] and BLAST-based taxonomic assignment in the QIIME environment [41], with run parameters established during prior in silico testing on trypanosomatid 18S rRNA sequences from NCBI. Samples were clustered to OTUs de novo at 98% sequence similarity and assigned to extant species with a confidence threshold of 80%. Unassigned clusters were considered valid OTUs only if present at > 300 reads in any single sample and present at > 600 reads across all samples of the dataset.
Following OTU establishment, sequence read pairs from one representative per OTU were merged and aligned in Clustal W (with manual refinement of misplaced reads). Phylogenies were inferred in Mega 6 [42] by maximum likelihood (ML) tree construction under Kimura’s two-parameter model of nucleotide substitution with gamma-distributed variation among sites (K2 + G). One thousand bootstrap replicates were run to establish nodal support. The 50 18S rRNA reference sequences applied in phylogenetic analyses are listed with accession numbers in S1 Table. All sequences have been deposited in the NCBI Sequence Read Archive (SRA) under accession numbers SRR5451077-SRR5451120.
Results
Bat abundance and diversity
Of the 108 bats captured at Amarelos, Buenos Aires and Rio da Prata study sites, 105 individuals represent 16 species in the Phyllostomidae family, and three individuals represent one species (Myotis nigricans) in the Vespertilionidae family. Species and their abundances are listed in Table 1.
Trypanosomatid abundance, diversity and distribution in bats
Standard and/or nested PCR amplified 18S rRNA gene fragments from 34 of 90 (38%) bat blood samples. The 34 positive samples derived from 13 bat species (of 17 species analysed) and comprised 14 distinct kinetoplastid OTUs. Five OTUs were assigned to T. cruzi I (OTU 3), T. cruzi III/IV (OTU 5), T. c. marinkellei (OTU 6), T. rangeli lineage D (OTU 10) and T. dionisii (OTU 2). A further seven OTUs did not assign to any known species of the T. cruzi clade. Phylogenetic analyses placed these seven OTUs (1, 7, 8, 11, 12, 13 and 14) within a monophyletic group that includes trypanosome species from bats of the New World. Finally, two OTUs showed greater homology outside of the T. cruzi clade—OTU 4, similar to a trypanosomatid species found in reptiles, and OTU 9, nearly identical to the eubodonid Bodo saltans (Figs 1 and 2, S1 Table).
Tree construction from 18S rRNA followed the maximum likelihood (ML) method under Kimura’s two-parameter model and gamma-distributed variation among sites (K2 + G). Numbers at nodes indicate support from 1000 bootstrap replicates. The 14 OTUs clustered into the T. cruzi clade (OTUs 1, 2, 3, 5, 6, 7, 8, 10, 11, 12, 13 and 14), a reptile-associated region (OTU 4) and the B. saltans outgroup (OTU 9).
Tree construction from 18S rRNA followed the maximum likelihood (ML) method under Kimura’s two-parameter model and gamma-distributed variation among sites (K2 + G). Numbers at nodes indicate support from 1000 bootstrap replicates.
Most trypanosome-infected bats presented mixed infections by two to eight OTUs. Only three positive blood samples (from D. rotundus, G. soricina and R. pumilio) contained a single OTU (T. dionisii; OTU 2). The bat species A. lituratus, C. perspicillata, D. rotundus and P. recifinus presented greatest trypanosome diversity, with seven to eight OTUs per species (Fig 3). Across the three study sites, trypanosomatid diversity and abundance broadly reflected bat capture success rather than any feature of the capture environment (Table 1 and Fig 4).
Each column represents the infection profile of one infected bat individual. Cell colour denotes the sequence read intensity attributed to each OTU (left), increasing from purple (zero reads) through yellow into red. Bat species and sample IDs are given above/below. Asterisks indicate samples subjected to nested PCR, of which ten also underwent standard PCR (dashed lines). Phylogenetic relationships inferred from 18S rRNA by maximum likelihood (ML) tree construction are plotted at right.
Sequence reads attributed to each OTU are color-coded by proportions obtained from bats captured at Amarelos (magenta), Buenos Aires (blue) and Rio da Prata (violet) study sites.
Standard vs. nested PCR sensitivity
Nested PCR detected between one and six more OTUs than standard PCR in eight of ten samples subjected to both procedures, showing less sensitivity only in samples RM 847 and RM 2009—one and two less OTUs amplified, respectively (Fig 3).
Discussion
In this study, we exposed unforeseen bat trypanosome 18S rRNA diversity from standard capture effort in Atlantic Forest fragments of Guarapari municipality, ES, southeast Brazil. Our metabarcoding approach identified a preponderance of coinfection, involving several human-pathogenic and bat-associated types of the T. cruzi clade, as well as a swathe of yet undescribed diversity closer to its base. Furthermore, we identified sequences from two divergent kinetoplastid taxa—one similar to trypanosomatid isolates from reptiles, another matching the non-parasitic B. saltans.
Unprecedented as they may be as complex co-infections, the diversity of individual kinetoplastids we report is not unexpected. Every recent trypanosome survey of bats has revealed novel parasite genotypes, host- and/or geographic range [8, 43–50], with particular surges in discovery following intensified sampling (e.g., transcontinental archival analysis) [8] or innovative approach (e.g., coalescent species delimitation) [43]. The 18S rRNA deep sequencing in bats here identifies further diversity around the most basal T. cruzi clade trypanosomes of the New World, with seven independent and novel taxonomic units forming sister groups to T. wauwau and Neobat species found in mormoopid and phyllostomid bats [8]. This expansion of a group related more closely to trypanosomatids detected in Australian marsupials than to those known from other neotropical mammals’ points to the Chiroptera as an ancient, perhaps original host order of the T. cruzi clade. Our data reinforce the bat host range of T. cruzi-clade trypanosomes across frugivorous, nectarivorous, carnivorous, generalist and hematophagous phyllostomid genera (Anoura, Artibeus, Carollia, Desmodus, Glossophaga, Platyrrhinus, Phyllostomus, Rhinophylla, Sturnira, Trachops) and into the (primarily insectivorous) Vespertilionidae.
Our study provides strong, if circumstantial, evidence for the role of bats as T. cruzi reservoirs in ES state. Trypanosoma cruzi I and III/V found in bats of this study correspond to Discrete Typing Units (DTUs) associated with a recent fatal T. cruzi–T. dionisii mixed infection and occur in Triatoma vitticeps at the study site [32]. These DTUs were not detected in parasitological or serological tests on local rodents and marsupials [32]. Triatoma vitticeps is thought to have poor stercocarian vector competence [51] and oral transmission via insectivory may be one of the few ways in which this species propagates disease. The apparent transfer of trypanosome diversity en masse from bat to human host via ingestion of the vector [32] supports transmission efficiency reported elsewhere in oral outbreaks [52]. Furthermore, given the low terrestrial mammal abundance in the heavily fragmented region where the samples were collected [32], bats may function here as principal reservoirs of parasites. There is growing evidence of bats’ potential in the maintenance of zoonotic T. cruzi transmission elsewhere in South America. For example, recent molecular surveys rank bats as top feeding sources of synanthropic T. cruzi-infected triatomines throughout Colombia, emphasize bats’ bridging of domestic and sylvatic transmission cycles in rural areas of Ecuador [45] (where non-volant hosts have shown limited infection [53–54]) and implicate bats as long-term refuges for parasites in areas subject to transmission interventions in Argentina [46]. Evidence of a new T. cruzi genotype associated with anthropogenic bats (TcBat) is also accumulating from around the continent [45, 55–58]. TcBat was not, however, observed in this study.
Here, we also provide first report of T. rangeli lineage D in bats, a strain initially isolated from Phyllomys dasythrix in southern Brazil [59]. As the ecogeographical structure of the Rhodnius spp. complex is thought to drive lineage divergence in T. rangeli [60–61], an efficiently transmitted salivarian parasite, our detection of lineage D further north and beyond the Rodentia serves well to confirm theory. Its putative vector R. domesticus [62] occurs throughout the Atlantic Forest, often in bromeliads [63] that rely on nectarivorous bats (e.g., the specialist flower-feeder A. caudifer) for pollination [64].
Whilst the expansion of the range of T. rangeli comes as little surprise, the presence of trypanosomes (OTU 4) with reptilian affinities in our study population is perhaps more intriguing. Nonetheless, bats and reptiles do commonly co-occur in an arboreal niche. Ecological host-fitting, involving opportunistic host switching mediated by vectors' feeding patterns within an ecological niche, is thought to be a prevailing mode of trypanosome evolution [65]. Reptilian trypanosomes are transmitted by sand fly vectors [65–66], with reports from Amazonia (Viannamyia tuberculate [67]) as well as central Brazil (Evandromyia evandroi [68]). Shared microhabitat use among bats, reptiles and sand flies potentiates spill-over of the parasite.
Most trypanosomatid diversity observed in this study was associated with complex mixed infections, a likely consequence of bats’ gregarious way of life. Tolerance of intracellular pathogens in the Chiroptera [21] suggests that multiple subclinical/asymptomatic infections may well accumulate in these hosts before triggering pathology linked to adaptive immune reactions in other non-volant mammals [69–72]. Frequent mixed infections, often coupled with low parasitaemia, have impeded bat trypanosome surveys in the past, both in genotyping from primary samples (e.g., low sensitivity in classic barcoding) [73] and on cultured cells (e.g., growth bias) [55, 61, 74]. The data presented here suggest that deep sequencing can resolve both infection identity and complexity.
Although our study demonstrates the power of the metabarcoding approach, several caveats are relevant. Sensitivity to contamination and errors from amplification and sequencing are of foremost concern [27, 75]. We employed a variety of cautionary measures during sample processing (e.g., flame-sterilized blood withdrawal, multiple negative DNA extraction/amplification controls) and in the bioinformatic phase: prior to taxonomic inference, we sent sequenced amplicons through a severe quality filter (99.9% base call accuracy), absorbed potential artefactual variance into broad 98% similarity clusters and rejected unassigned OTUs present at low to moderate depth (< 300/600 reads). Nevertheless, our study would have benefited from the inclusion of traditional methods (e.g., microscopy, ex and in vivo culture) for validation and follow-up. Based on subunit rRNA, OTU 9, isolated from a single bat (D. rotundus), was assigned to B. saltans, considered the closest free-living relative of the parasitic trypanosomatids. This observation joins others in unsettling assumptions about putatively free-living, yet seldom studied protist taxa. For example, 18S rRNA analysis (complemented by microscopy and serological testing) found an apparent case of babesiosis in China to involve erythrocytic colpodellids, the closest “free-living” relatives of the parasitic Apicomplexa [76].
Regrettably, our field-based study passed over visual and biochemical tests that could have established the occurrence and viability of OTU 9 in mammalian tissue and we hesitate to entirely rule out environmental contamination as its source. Bodo saltans belongs to the most widely adapted, physiologically tolerant zooflagellates on Earth [77]. It abounds in soil and water and can also spread in aerosolized forms. As such, this eubodonid may in rare cases happen upon sampling equipment as well as resist certain antiseptic measures taken in the field. On account of its exceptional halotolerance [78], for example, B. saltans may withstand some iodine-based disinfection (as do other protozoans—e.g., Cryptosporidium and Giardia [79]), though very unlikely as performed in this study (i.e., with ethanol). More importantly, however, OTU detection does not require a living organism, only its DNA. Severe contamination from the “dead” DNA of protist flagellates has indeed preoccupied past rRNA sequence analysis (e.g., see methods in [80]). In any case, we suggest additional (environmental) control samples (e.g., vials opened in the field, topical swabs around the site of cardiac puncture) and laboratory efforts that distinguish DNA from viable cells (e.g., separation of lysed and non-lysed cells, RNA/DNA comparisons) to help test for such possibilities in future research.
In this section of Atlantic Forest, where a rural Chagas disease fatality in all likelihood involved a bat-feeding triatomine [32], our deep sequencing study highlights the role of the Chiroptera as a reservoir for trypanosomiases. Furthermore, the unprecedented transfer of T. dionisii to a human from a bat, as well as the presence of reptile-infecting and putatively non-parasitic kinetoplastids in the same bat population, highlights the role of bats as keystone species in parasite spill-over events. Many questions remain on how the role of sylvatic hosts in pathogen dispersal varies in space and time, upon change to environment and at the evolutionary scale. Research into these intricacies of complex zoonosis will require much further innovation with high-sensitivity, high-throughput tools. We point to the power of next-generation metabarcoding strategies in studies of trypanosomatid ecology and evolution and strongly commend their future complementation with non-molecular methods.
Supporting information
S1 Fig. Representative map of bat capture locations in Atlantic Forest of Guarapari municipality, ES state, Brazil.
https://doi.org/10.1371/journal.pntd.0005790.s001
(TIF)
S1 Table. GenBank reference sequences used in phylogenetic analyses of kinetoplastid 18S rRNA.
https://doi.org/10.1371/journal.pntd.0005790.s002
(DOCX)
Acknowledgments
We would like to thank Luciana M. Costa (UERJ), Bruno Alves (IOC/Fiocruz) and Roberto Leonan Morim Novaes (UFRJ) for fieldwork support. Thanks to Alcidelio Lovatti, Helton Meriguete and Pricila Pietralonga from Zoonoses Control Center, Guarapari municipality, for laboratory support during fieldwork. Thanks to Michele Giovannino Tancredi for various laboratory assistance. Thanks to Julie Galbraith at Glasgow Polyomics for assistance with amplicon library preparation.
References
- 1. World Health Organization. Chagas Disease in Latin America: an epidemiological update based on 2010 estimates. Wkly Epidemiol Rec. 2015;90: 33–43. pmid:25671846
- 2. Hamilton PB, Stevens JR, Gaunt MW, Gidley J, Gibson WC. Trypanosomes are monophyletic: evidence from genes for glyceraldehyde phosphate dehydrogenase and small subunit ribosomal RNA. Int J Parasitol. 2004;34: 1393–1404. pmid:15542100
- 3. Lima L, Espinosa-Álvarez O, Hamilton PB, Neves L, Takata CSA, Campaner M, et al. Trypanosoma livingstonei: a new species from African bats supports the bat seeding hypothesis for the Trypanosoma cruzi clade. Parasit Vectors. 2013;6(1): 221. pmid:23915781
- 4. Hamilton PB, Adams ER, Njiokou F, Gibson WC, Cuny G, Herder S. Phylogenetic analysis reveals the presence of the Trypanosoma cruzi clade in African terrestrial mammals. Infect Genet Evol. 2009;9: 81–6. pmid:19027884
- 5. Noyes HA, Stevens JR, Teixeira MMGT, Phelan J, Holz P. A nested PCR for the ssrRNA gene detects Trypanosoma binneyi in platypus and Trypanosoma sp. in wombats and kangaroos in Australia. Int J Parasitol. 1999;29: 331–9. pmid:10221634
- 6. Jansen AM, Xavier SC, Roque AL. The multiple and complex and changeable scenarios of the Trypanosoma cruzi transmission cycle in the sylvatic environment. Acta Trop. 2015;151: 1–15. pmid:26200785
- 7. Lima L, Maia da Silva F, Neves L, Attias M, Takata CS, Campaner M, et al. Evolutionary insights from bat trypanosomes: morphological, developmental and phylogenetic evidence of a new species, Trypanosoma (Schizotrypanum) erneyi sp. nov. in African bats closely related to Trypanosoma (Schizotrypanum) cruzi and allied species. Protist. 2012;163: 856–72. pmid:22277804
- 8. Lima L, Espinosa-Álvarez O, Pinto CM, Cavazzana M Jr, Pavan AC, Carranza JC, et al. New insights into the evolution of the Trypanosoma cruzi clade provided by a new trypanosome species tightly linked to Neotropical Pteronotus bats and related to an Australian lineage of trypanosomes. Parasit Vectors. 2015;8: 657. pmid:26701154
- 9. Zhang G, Cowled C, Shi Z, Huang Z, Bishop-Lilly KA, Fang X, et al. Comparative analysis of bat genomes provides insight into the evolution of flight and immunity. Science. 2013;339(6118): 456–460. pmid:23258410
- 10. O’Shea TJ, Cryan PM, Cunningham AA, Fooks AR, Hayman DTS, Luis AD, et al. Bat flight and zoonotic viruses. Emer Infect Dis. 2014;20: 741–5.
- 11. Carini A. Sur une grande épizootie de range. Ann Inst Pasteur (Paris).1911;25: 843–6.
- 12. Turmelle AS, Jackson FR, Green D, McCracken GF, Rupprecht CE. Host immunity to repeated rabies virus infection in big brown bats. J Gen Virol. 2010;91: 2360–6 pmid:20519458
- 13. Kosoy M, Bai Y, Lynch T, Kuzmin IV, Niezgoda M, Franka R, et al. Bartonella spp. in bats, Kenya. Emerg Infect Dis. 2010;16: 1875–81. pmid:21122216
- 14. Evans NJ, Brown K, Timofte D, Simpson VR, Birtles RJ. Fatal borreliosos in bat caused by relapsing fever spirochete, United Kingdom. Emerg Infect Dis. 2009;15: 1330–1.
- 15. Greer DL, McMurray AN. Pathogenesis of experimental histoplasmosis in the bat, Artibeus lituratus. Am J Trop Med Hyg. 1981;30: 653–9. pmid:7258485
- 16. Meteyer CU, Barber D, Mandi JN. Pathology in euthermic bats with white nose syndrome suggests a natural manifestation of immune reconstitution inflammatory syndrome. Virulence. 2012;3: 1–6.
- 17. Hamilton PB, Teixeira MM, Stevens JR. The evolution of Trypanosoma cruzi: the “bat seeding” hypothesis. Trends Parasitol. 2012;28: 136–41. pmid:22365905
- 18. Schaer J, Perkins SL, Decher J, Leendertz FH, Fahr J, Weber N, et al. High diversity of West African bat malaria parasites and a tight link with rodent Plasmodium taxa. Proc Natl Acad Sci USA. 2013;110: 17415–9. pmid:24101466
- 19. Lichtenfels JR, Bhatnagar KP, Frahm HD. Filaroid nematodes in olfactory mucosa, olfactory bulb and brain ventricular ventricular system of bats. Trans Am Microsc Soc. 1981;100: 216–9.
- 20.
Ubelaker JE. Some observations on ecto and endoparasites of Chiroptera. In: Slaughter BH, Walton DW, editors. About Bats. Dallas: Southern Methodist University Press; 1970. pp. 247–61.
- 21. Brook CE, Dobson AP. Bats as ‘special’ reservoirs for emerging zoonotic pathogens. Trends Microbiol. 2015;23: 172–80. pmid:25572882
- 22. Stevens JR, Gibson W. The molecular evolution of trypanosomes. Parasitol Today. 1999;15: 432–7. pmid:10511684
- 23.
Hoare CA. The trypanosomes of mammals: a zoological monograph. Oxford: Blackwell Scientific Publications; 1972.
- 24. Kircher M, Kelso J. High-throughput DNA sequencing—concepts and limitations. BioEssays. 2010;32: 524–36. pmid:20486139
- 25. Glenn TC. Field guide to next-generation DNA sequencers. Mol Ecol Resour. 2011;11: 759–69. pmid:21592312
- 26. Pompanon F, Deagle BE, Symondson WOC, Brown DS, Jarman SN, Taberlet P. Who is eating what: diet assessment using next generation sequencing. Mol Ecol. 2012;21(8): 1931–50. pmid:22171763
- 27. Taberlet P, Coissac E, Pompanon F, Brochmann C, Willerslev E. Towards next-generation biodiversity assessment using DNA metabarcoding. Mol Ecol. 2012;21: 2045–50. pmid:22486824
- 28. Liu W, Learn GH, Rudicell RS, Robertson JD, Keele BF, Ndjango JBN, et al. Origin of the human malaria parasite Plasmodium falciparum in gorillas. Nature. 2010;467: 420–5. pmid:20864995
- 29. Ji Y, Ashton L, Pedley SM, Edwards DP, Tang Y, Nakamura A, et al. Reliable, verifiable and efficient monitoring of biodiversity via metabarcoding. Ecol Lett. 2013;16: 1245–57. pmid:23910579
- 30. Pagenkopp-Lohan KM, Fleischer RC, Carney KJ, Holzer KK, Ruiz GM. Amplicon-based pyrosequencing reveals high diversity of protistan parasites in ships’ ballast water: implications for biogeography and infectious diseases. Microbial Ecol. 2016;71: 530–42.
- 31.
Fonseca GAB, Herrmann G, Leite YLR. Macrogeography of Brazilian mammals. In: Eisenberg JF, Redford KH, editors. Mammals of the Neotropics. The Central Neotropics: Ecuador, Peru, Bolivia, Brazil. Chicago: University of Chicago Press; 1999. pp. 549–63.
- 32. Dario MA, Rodrigues MS, Barros JH, Xavier SC, D'Andrea PS, Roque AL, et al. Ecological scenario and Trypanosoma cruzi DTU characterization of a fatal acute Chagas disease case transmitted orally (Espírito Santo state, Brazil). Parasit Vectors. 2016;9: 477. pmid:27580853
- 33.
Gardner AL. Mammals of South America: marsupials, xenarthrans, shrews, and bats. Chicago: University of Chicago Press; 2008.
- 34. Smith A, Clark P, Averis S, Lymbery AJ, Wayne AF, Morris KD. et al. Trypanosomes in a declining species of threatened Australian marsupial, the brush-tailed bettong Bettongia penicillata (Marsupialia: Potoroidae). Parasitology. 2008;135: 1329–35. pmid:18752704
- 35. Zhou J, Wu L, Deng Y, Zhi X, Jiang Y-H, Tu Q, et al. Reproducibility and quantitation of amplicon sequencing-based detection. ISME J. 2011;5: 1303–13. pmid:21346791
- 36.
Joshi N, Fass J. Sickle: A sliding-window, adaptive, quality-based trimming tool for FastQ files (Version 1.29) [Software]. 2011.
- 37.
Andrews S. FastQC: a quality control tool for high throughput sequence data (Version 0.11.5, 2016). http://www.bioinformatics.babraham.ac.uk/projects/fastqc.
- 38. Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucl Acids Res. 2013;41(D1): 590–6.
- 39. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012; 9: 357–9. pmid:22388286
- 40. Edgar RC. UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat Methods. 2013;10: 996–8. pmid:23955772
- 41. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, et al. QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 2010;7: 335–6. pmid:20383131
- 42. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol Biol Evol. 2013;30(12): 2725–9. pmid:24132122
- 43. Cottontail VM, Kalko EKV, Cottontail I, Wellinghausen N, Tschapka M, Perkins SL, et al. High local diversity of Trypanosoma in a common bat species, and implications for the biogeography and taxonomy of the T. cruzi clade. PLoS One. 2014;9(9): e108603. pmid:25268381
- 44. Austen JM, O’Dea M, Jackson B, Ryan U. High prevalence of Trypanosoma vegrandis in bats Western Australia. Vet Parasitol. 2015;214: 342–7. pmid:26541211
- 45. Pinto CM, Ocaña-Mayorga S, Tapia EE, Lobos SE, Zurita AP, Aguirre-Villacís F, et al. Trypanosomes, and Triatomines in Ecuador: New Insights into the Diversity, Transmission, and Origins of Trypanosoma cruzi and Chagas Disease. PLoS One. 2015;10(10): e0139999. pmid:26465748
- 46. Argibay HD, Orozco MM, Cardinal MV, Rinas MA, Arnaiz M, Mena Segura C, et al. First finding of Trypanosoma cruzi II in vampire bats from a district free of domestic vector-borne transmission in Northeastern Argentina. Parasitology. 2016;143(11):1358–68. pmid:27220254
- 47. Barbosa AD, Mackie JT, Stenner R, Gillett A, Irwin P, Ryan U. Trypanosoma teixeirae: a new species belonging to the T. cruzi clade causing trypanosomiasis in an Australian little red flying fox (Pteropus scapulatus). Vet Parasitol. 2016;223: 214–21. pmid:27198803
- 48. da Costa AP, Nunes PH, Leite BH, Ferreira JI, Tonhosolo R, da Rosa AR, et al. Diversity of bats trypanosomes in hydroeletric area of Belo Monte in Brazilian Amazonia. Acta Trop. 2016;164: 185–93. pmid:27633579
- 49. Hodo CL, Goodwin CC, Mayes BC, Mariscal JA, Waldrup KA, Hamer SA. Trypanosome species, including Trypanosoma cruzi, in sylvatic and peridomestic bats of Texas, USA. Acta Trop. 2016;164: 259–66. pmid:27647574
- 50. Orozco MM, Enriquez GF, Cardinal MV, Piccinali RV, Gürtler RE. A comparative study of Trypanosoma cruzi infection in sylvatic mammals from a protected and a disturbed area in the Argentine Chaco. Acta Trop. 2016;155: 34–42. pmid:26708994
- 51. dos Santos CB, Leite GR, Sessa PA, Falqueto A. Dynamics of feeding and defecation in Triatoma vitticeps (Stal, 1859) (Hemiptera, Reduviidae, Triatominae) and its potential in the transmission of Trypanosoma cruzi. Mem Inst Oswaldo Cruz. 2006;101(5): 543–6. pmid:17072459
- 52. Segovia M, Carrasco HJ, Martínez CE, Messenger LA, Nessi A, Londoño JC, et al. Molecular epidemiologic source tracking of orally transmitted Chagas disease, Venezuela. Emerg Infect Dis. 2013;19(7): 1098–1101. pmid:23768982
- 53. Pinto CM, Grijalva MJ, Costales JA. Prevalencia de Trypanosoma cruzi en roedores y marsupiales en dos localidades de Manabí, Ecuador. Rev Pontif Univ Católica Ecuad. 2003;71: 225–33.
- 54. Pinto CM, Ocaña-Mayorga S, Lascano MS, Grijalva MJ. Infection by trypanosomes in marsupials and rodents associated with human dwellings in Ecuador. J Parasitol. 2006;92: 1251–5. pmid:17304802
- 55. Marcili A, Lima L, Cavazzana M, Junqueira AC, Veludo HH, Maia Da Silva F, et al. A new genotype of Trypanosoma cruzi associated with bats evidenced by phylogenetic analyses using SSU rDNA, cytochrome b and Histone H2B genes and genotyping based on ITS1 rDNA. Parasitology. 2009;136(6): 641–55. pmid:19368741
- 56. Pinto CM, Kalko EK, Cottontail I, Wellinghausen N, Cottontail VM. TcBat a bat-exclusive lineage of Trypanosoma cruzi in the Panama Canal Zone, with comments on its classification and the use of the 18S rRNA gene for lineage identification. Infect Genet Evol. 2012;12(6): 1328–32. pmid:22543008
- 57. Ramírez JD, Tapia-Calle G, Muñoz-Cruz G, Poveda C, Rendón LM, Hincapié E, et al. Trypanosome species in neo-tropical bats: biological, evolutionary and epidemiological implications. Infect Genet Evol. 2014;22: 250–6. pmid:23831017
- 58. Cura CI, Duffy T, Lucero RH, Bisio M, Péneau J, Jimenez-Coello M, et al. Multiplex real-time PCR assay using TaqMan probes for the identification of Trypanosoma cruzi DTUs in biological and clinical samples. PLoS Negl Trop Dis. 2015;9(5): e0003765. pmid:25993316
- 59. Steindel M, Carvalho Pinto JC, Toma HK, Mangia RHR, Ribeiro-Rodrigues R, Romanha AJ. Trypanosoma rangeli (Tejera, 1920) isolated from a sylvatic rodent (Echimys dasythrix) in Santa Catarina Island, Santa Catarina State: first report of this trypanosome in southern Brazil. Mem Inst Oswaldo Cruz. 1991;86(1): 73–9. pmid:1842404
- 60. Maia Da Silva F, Junqueira AC, Campaner M, Rodrigues AC, Crisante G, Ramirez LE, et al. Comparative phylogeography of Trypanosoma rangeli and Rhodnius (Hemiptera: Reduviidae) supports a long coexistence of parasite lineages and their sympatric vectors. Mol Ecol. 2007;16(16): 3361–73. pmid:17688539
- 61. Maia da Silva F, Marcili A, Lima L, Cavazzana M Jr, Ortiz PA, Campaner M, et al. Trypanosoma rangeli isolates of bats from Central Brazil: genotyping and phylogenetic analysis enable description of a new lineage using spliced-leader gene sequences. Acta Trop. 2009;109: 199–207. pmid:19063857
- 62. Steindel M, Dias Neto E, Pinto CJ, Grisard EC, Menezes CL, Murta SM, Simpson AJ, Romanha AJ. Randomly amplified polymorphic DNA (RAPD) and isoenzyme analysis of Trypanosoma rangeli strains. J Eukaryot Microbiol. 1994;41(3): 261–7. pmid:8049688
- 63. Abad-Franch F, Palomeque FS, Aguilar HM, Miles MA. Field ecology of sylvatic Rhodnius populations (Heteroptera, Triatominae): risk factors for palm tree infestation in western Ecuador. Trop Med Int Health. 2005;10(12): 1258–66. pmid:16359406
- 64. Fleming TH, Geiselman C, Kress WJ. The evolution of bat pollination: a phylogenetic perspective. Ann Bot. 2009;104(6): 1017–43. pmid:19789175
- 65. Hamilton PB, Gibson WC, Stevens JR. Patterns of coevolution between trypanosomes and their hosts deduced from ribosomal RNA and protein-coding gene phylogenies. Mol Phylog Evol. 2007;44: 15–25.
- 66.
Telford RS. The kinetoplastid hemoflagellates of reptiles. In: Kreier JP editor. Parasitic Protozoa. 2nd ed. New York: Academic Press; 1995;10. pp. 161–223.
- 67. Viola LB, Campaner M, Takata CSA, Ferreira RC, Rodrigues AC, Freitas RA, et al. Phylogeny of snake trypanosomes inferred by SSU rRNA sequences, their possible transmission by phlebotomines, and taxonomic appraisal by molecular, cross-infection and morphological analysis. Parasitology. 2008;135: 595–605. pmid:18371240
- 68. Ferreira ST, Minuzzi-Souza TT, Andrade AJ, Coelho TO, Rocha Dde A, Obara MT, et al. Molecular detection of Trypanosoma sp. and Blastocrithidia sp. (Trypanosomatidae) in phlebotomine sand flies (Psychodidae) in the Federal District of Brazil. Rev. Soc. Bras. Med. Trop. 2015;48(6): 776–9. pmid:26676507
- 69. Andrade ZA. Immunopathology of Chagas disease. Mem Inst Oswaldo Cruz. 1999;94(S1): 71–80.
- 70. Perez CJ, Lymbery AJ, Thompson RC. Chagas disease: the challenge of polyparasitism? Trends Parasitol. 2014;30(4): 176–82. pmid:24581558
- 71. Rodrigues CM, Valadares HM, Francisco AF, Arantes JM, Campos CF, Teixeira-Carvalho A, et al. Coinfection with different Trypanosoma cruzi strains interferes with the host immune response to infection. PLoS Negl Trop Dis. 2010;4(10): e846. pmid:20967289
- 72. Galán-Puchades M, Osuna A. Chagas disease in a wormy world. Rev Ibero-Latinoam Parasitol. 2012;71: 5–13.
- 73. Marcili A, da Costa AP, Soares HS, Acosta IaC, de Lima JT, Minervino AH, Melo , et al. Isolation and phylogenetic relationships of bat trypanosomes from different biomes in Mato Grosso, Brazil. J. Parasitol. 2013;99: 1071–6. pmid:23859496
- 74. Cavazzana M Jr, Marcili A, Lima L, da Silva FM, Junqueira AC, Veludo HH, et al. Phylogeographical, ecological and biological patterns shown by nuclear (ssrRNA and gGAPDH) and mitochondrial (Cyt b) genes of trypanosomes of the subgenus Schizotrypanum parasitic in Brazilian bats. Int J Parasitol. 2010;40: 345–55. pmid:19766649
- 75. Coissac E, Riaz T, Puillandre N. Bioinformatic challenges for DNA metabarcoding of plants and animals. Mol Ecol. 2012;21(8): 1834–47. pmid:22486822
- 76. Yuan CL, Keeling PJ, Krause PJ, Horak A, Bent S, Rollend L, Hua XG. Colpodella spp.–like parasite infection in woman, China. Emerg Infect Dis. 2012;18(1): 125–7. pmid:22260904
- 77. Lee WJ, Patterson DJ. Diversity and geographic distribution of free-living heterotrophic flagellates—Analysis by PRIMER. Protist. 1998;149(3): 229–44. pmid:23194636
- 78.
Hauer G., Rogerson A. (2005) Heterotrophic protozoa from hypersaline environments. In: Gunde-Cimerman N., Oren A., Plemenitaš A., editors. Adaptation to life at high salt concentrations in Archaea, Bacteria, and Eukarya. Cellular origin, life in extreme habitats and astrobiology, vol. 9. Dordrecht: Springer; 2005. pp. 519–39.
- 79. McDonnell G, Russell AD. Antiseptics and disinfectants: activity, action, and resistance. Clin Microbiol Rev. 1999;12(1): 147–79. pmid:9880479
- 80. Auinger BM, Pfandl K, Boenigk J. Improved methodology for identification of protists and microalgae from plankton samples preserved in lugol’s iodine solution: combining microscopic analysis with single-cell PCR. Appl Environ Microbiol. 2008;74(8): 2505–10. pmid:18296536