Small subunit ribosomal metabarcoding reveals extraordinary trypanosomatid diversity in Brazilian bats

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

High Trypanosoma spp. diversity is maintained by bats and triatomines in Espírito Santo state, Brazil

The aim of this study was to reevaluate the ecology of an area in the Atlantic Forest, southeast Brazil, where Chagas disease (CD) has been found to occur. In a previous study, immediately after the occurrence of a CD case, we did not observe any sylvatic small mammals or dogs with Trypanosoma cruzi cruzi infections, but Triatoma vitticeps presented high T. c. cruzi infection rates. In this study, we investigated bats together with non-volant mammals, dogs, and triatomines to explore other possible T. c. cruzi reservoirs/hosts in the area. Seventy-three non-volant mammals and 186 bats were captured at three sites within the Guarapari municipality, Espírito Santo state. Rio da Prata and Amarelos sites exhibited greater richness in terms of non-volant mammals and bats species, respectively. The marsupial Metachirus nudicaudatus, the rodent Trinomys paratus, and the bats Artibeus lituratus and Carollia perspicillata were the most frequently captured species. As determined by positive hemocultures, only two non-volant mammals were found to be infected by Trypanosoma species: Monodelphis americana, which was infected by T. cascavelli, T. dionisii and Trypanosoma sp., and Callithrix geoffroyi, which was infected by T. minasense. Bats presented T. c. cruzi TcI and TcIII/V, T. c. marinkellei, T. dionisii, T. rangeli B and D, and Trypanosoma sp. infections. Seven dogs were infected with T. cruzi based only on serological exams. The triatomines T. vitticeps and Panstrongylus geniculatus were found to be infected by trypanosomes via microscopy. According to molecular characterization, T. vitticeps specimens were infected with T. c. cruzi TcI, TcII, TcIII/V, and TcIV, T. c. marinkellei and T. dionisii. We observed high trypanosome diversity in a small and fragmented region of the Atlantic Forest. This diversity was primarily maintained by bats and T. vitticeps. Our findings show that the host specificity of the Trypanosoma genus should be thoroughly reviewed. In addition, our data show that CD cases can occur without an enzootic cycle near residential areas

Ecological scenario and Trypanosoma cruzi DTU characterization of a fatal acute Chagas disease case transmitted orally (Espírito Santo state, Brazil)

Submitted by Sandra Infurna ([email protected]) on 2016-12-09T13:37:11Z No. of bitstreams: 1 samanta_xavier_etal_IOC_2016.pdf: 1232916 bytes, checksum: cb48d61d779f9c21eabb66900de216bd (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2016-12-09T13:49:04Z (GMT) No. of bitstreams: 1 samanta_xavier_etal_IOC_2016.pdf: 1232916 bytes, checksum: cb48d61d779f9c21eabb66900de216bd (MD5)Made available in DSpace on 2016-12-09T13:49:04Z (GMT). No. of bitstreams: 1 samanta_xavier_etal_IOC_2016.pdf: 1232916 bytes, checksum: cb48d61d779f9c21eabb66900de216bd (MD5) Previous issue date: 2016Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia de Tripanossomatídeos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia de Tripanossomatídeos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia de Tripanossomatídeos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia de Tripanossomatídeos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia e Parasitologia de Mamíferos Silvestres Reservatórios. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia de Tripanossomatídeos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia de Tripanossomatídeos. Rio de Janeiro, RJ, Brasil.Trypanosoma cruzi infection via oral route results in outbreaks or cases of acute Chagas disease (ACD) in different Brazilian regions and poses a novel epidemiological scenario. In the Espírito Santo state (southeastern Brazil), a fatal case of a patient with ACD led us to investigate the enzootic scenario to avoid the development of new cases. At the studied locality, Triatoma vitticeps exhibited high T. cruzi infection rates and frequently invaded residences

Environmental DNA: a new low-cost monitoring tool for pathogens in salmonid aquaculture

Environmental DNA (eDNA) metabarcoding is a relatively new monitoring tool featuring in an increasing number of applications such as the facilitation of the accurate and cost effective detection of species in environmental samples. eDNA monitoring is likely to have a major impact on the ability of salmonid aquaculture industry producers and their regulators to detect the presence and abundance of pathogens and other biological threats in the surrounding environment. However, for eDNA metabarcoding to develop into a useful bio-monitoring tool it is necessary to (a) validate that sequence datasets derived from amplification of metabarcoding markers reflect the true species’ identity, (b) test the sensitivity under different abundance levels and environmental noise and (c) establish a low-cost sequencing method to enable the bulk processing of field samples. In this study, we employed an elaborate experimental design whereby different combinations of five biological agents were crossed at three abundance levels and exposed to sterile pre-filtered and unfiltered seawater, prior to coarse filtering and then eDNA ultrafiltration of the resultant material. We then benchmarked the low-cost, scalable, Ion Torrent sequencing method against the current gold-standard Illumina platform for eDNA surveys in aquaculture. Based on amplicon-seq of the 18S SSU rDNA v9 region, we were able to identify two parasites (Lepeophtheirus salmonis and Paramoeba perurans) to species level, whereas the microalgae species Prymnesium parvum, Pseudo-nitzschia seriata, and P. delicatissima could be assigned correctly only to the genus level. Illumina and Ion Torrent provided near identical results in terms of community composition in our samples, whereas Ion Torrent was more sensitive in detecting species richness when the medium was unfiltered seawater. Both methods were able to reflect the difference in relative abundance between treatments in 4 out of 5 species when samples were exposed to the unfiltered seawater, despite the significant amount of background noise from both bacteria and eukaryotes. Our findings indicate that eDNA metabarcoding offers significant potential in the monitoring of species harmful to aquaculture and for this purpose, the low-cost Ion Torrent sequencing is as accurate as Illumina in determining differences in their relative abundance between samples

Bat species captured in Guarapari municipality, ES state, Brazil.

<p>Bat species captured in Guarapari municipality, ES state, Brazil.</p

Heatmap of kinetoplastid OTU distribution among bats captured in Guarapari municipality, ES state, Brazil.

<p>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.</p

Phylogenetic placement of kinetoplastid OTUs detected in bats of Guarapari municipality, ES state, Brazil.

<p>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 <i>T</i>. <i>cruzi</i> clade (OTUs 1, 2, 3, 5, 6, 7, 8, 10, 11, 12, 13 and 14), a reptile-associated region (OTU 4) and the <i>B</i>. <i>saltans</i> outgroup (OTU 9).</p