Detection of Trypanosoma cruzi in the saliva of diverse neotropical bats

Abstract Trypanosoma cruzi is widely reported in bats, yet transmission routes remain unclear. We present evidence from metagenomic sequence data that T. cruzi occurs in the saliva of diverse Neotropical bats. Phylogenetic analyses demonstrated that the bat‐associated T. cruzi sequences described here formed part of a bat‐specific clade, suggesting an independent transmission cycle. Our results highlight the value in repurposing metagenomic data generated for viral discovery to reveal insights into the biology of other parasites. Evaluating whether the presence of T. cruzi in the saliva of two hematophagous bat species represents an ecological route for zoonotic transmission of Chagas disease is an interesting avenue for future research.

predominately arise in domestic or peridomestic cycles of stercorarian transmission from triatomine vectors; however, alternative transmission routes of T. cruzi can include transfusion and transplantation (Bern, 2015;Perez-Molina & Molina, 2018). In light of successful vector control programs and serological screening in blood banks to prevent transfusions of infected blood, congenital transmission and orally transmitted infections originating from sylvatic cycles are of increasing epidemiological importance (Perez-Molina & Molina, 2018;Shikanai-Yasuda & Carvalho, 2012). Here, we focus on sylvatic cycles of T. cruzi in wildlife, which can be maintained in animal populations through vector-borne transmission, consumption of contaminated material, or predation on infected hosts or vectors (Jansen et al., 2015). Additionally, some wildlife species such as opossums experimentally and naturally maintain multiple parasite life stages (Barros et al., 2020;Deane et al., 1984) and have been hypothesized to transmit T. cruzi in the absence of arthropod vectors (Shikanai-Yasuda et al., 1991;Urdaneta-Morales & Nironi, 1996).
The recent detection of T. cruzi in the salivary glands of Diaemus youngi, a hematophagous bat, suggests the possibility that bats could also act as both reservoirs and transmitters of the parasite (Villena et al., 2018). Bats are important trypanosome reservoirs which host both generalist and bat-restricted trypanosomes (Marcili et al., 2009;Ramírez et al., 2014) and have been suggested as the ancestral host of trypanosomes (Hamilton et al., 2012). Identifying routes of trypanosome transmission in bats may shed new light on sylvatic cycles of the parasite and inform strategies to reduce zoonotic transmission.

| MATERIAL S AND ME THODS
As part of a virus discovery project, in 2016, we captured bats across seven sites in northern Peru (Departments of Amazonas, Cajamarca and Loreto) using mist nets, harp traps and hand nets ( Figure 1) (Bergner et al., 2020). Samples were collected from four bat species Saliva was collected using sterile cotton-tipped swabs (Fisherbrand) which were stored in 1ml RNALater (Ambion) overnight at 4°C then transferred to −80°C. Total nucleic acid was extracted from individual swabs using a KingFisher Flex 96 (Thermo) and a BioSprint One for All Vet Kit (Qiagen) (Bergner et al., 2019).
Extracts were pooled by bat species (Table 1) and depleted of host material using DNAse (Bergner et al., 2019). Libraries were prepared for untargeted metagenomic sequencing using the Clontech SMARTer Stranded Total RNA-Seq Kit v2 (Takara), then sequenced on an Illumina NextSeq500 at the University of Glasgow Polyomics Facility. Sequencing reads (European Nucleotide Archive project PRJEB35111) were processed using an in-house bioinformatic pipeline (Bergner et al., 2019), with slight modification to the read trimming step to accommodate the library preparation kit and read length.
cruzi cytB and gGAPDH sequences from different hosts and vectors (Table S1 and Table S2)  For each alignment, the best model of sequence evolution and support for codon partitioning were evaluated using PartitionFinder2 (Lanfear et al., 2017) on the CIPRES Science Gateway 3.3, which was run with linked branch lengths, the greedy search algorithm, and BIC criterion. For the cytB analysis, PartitionFinder supported codon partitioning with the models HKY + G, F81 and GTR + G applied to the first, second and third codon positions, respectively.  (Schliep, 2010), 'phytools' (Revell, 2011) and 'ggtree' (Yu et al., 2016).    (Figure 2; Figure S1; Figure S2). Although the Peruvian batderived sequences did not group together in the gGAPDH phylogeny, likely due to lack of sequence variation, cytB sequences clustered with TcI sequences from Brazilian bats (Lima et al., 2014) (posterior probability = 0.77; bootstrap support = 58%). Other

| RE SULTS AND D ISCUSS I ON
Neotropical bat-derived TcI sequences from Venezuela, Colombia and Brazil were dispersed amongst non-bat TcI samples or formed a distinct bat-associated clade towards the base of the TcI lineage ( Figure 2; Figure S1), as observed previously (Marcili et al., 2009).
Sequences from bat and non-bat hosts did not cluster together for any country where both were available (i.e., Venezuela, Colombia, Brazil), demonstrating that geographic structure alone does not explain the occurrence of bat-associated TcI clades (Table S1). TcI has been hypothesized to have its origins in marsupials due to high levels of strain diversity in these hosts (Brenière et al., 2016), but it also occurs in diverse bat species (Lima et al., 2014;Marcili et al., 2009;Ramírez et al., 2014). Our results support the conclusion that bats can maintain independent transmission cycles of this lineage. Although our approach focused only on TcI, future studies could employ metabarcoding (e.g., Dario et al., 2017) to explore the diversity of other Trypanosoma species present in bat saliva. More generally, as our data were originally generated for virus discovery, we show how metagenomic data can simultaneously reveal insights into diverse pathogens.
The discovery of T. cruzi in bat saliva has several plausible ecolog- replicate and transmit other Trypanosoma species (Gardner & Molyneux, 1988). Oral infection of humans by a similar route further supports the viability of this transmission mode (Shikanai-Yasuda & Carvalho, 2012). Alternatively, T. cruzi may be excreted in bat saliva, as supported by infection in the salivary glands of another hematophagous bat species, D. youngi (Villena et al., 2018). If verified, batto-bat transmission in the absence of arthropods would represent a novel transmission route which might occur through social contacts, biting, or-in the case of D. rotundus-blood-meal sharing.
Although T. cruzi has been documented in the salivary glands of D. youngi (Villena et al., 2018), our findings comprise the first evidence of TcI in the saliva of D. rotundus and D. ecaudata, two vampire bat species which are known to feed on humans (Ito et al., 2016).
Notably, the area of northern Peru where our study was conducted is a hotspot for vampire bat depredation on humans which has been associated with recurrent rabies outbreaks (Gilbert et al., 2012;Stoner-Duncan et al., 2014 Zoonotic transmission also depends on the susceptibility of humans to bat-associated strains. In our study, the cytB and gGAPDH phylogenies suggest that the parasites detected in bats belong to the TcI lineage of T. cruzi, which is generally assumed to be capable of infecting humans. However, we note that multi-locus sequence typing and 18S ribosomal RNA sequencing can more sensitively discriminate T. cruzi lineages, so additional sequencing of these markers is needed to confirm the identity of trypanosomes as T. cruzi I (Dario et al., 2017;Yeo et al., 2011). This is particularly relevant given that our sequences represent a consensus based on pools made up of multiple individuals, and others have reported a high frequency of mixed infections even in individuals bats (Dario et al., 2017;Jansen et al., 2018).
In conclusion, our study reports likely bat-maintained transmis-

ACK N OWLED G EM ENTS
We thank Philipp Schwabl, Diana Meza and Nicole Gottdenker for helpful discussions.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

DATA AVA I L A B I L I T Y S TAT E M E N T
Metagenomic sequence data are available on the European Nucleotide Archive (Project PRJEB35111 https://www.ebi.ac.uk/ ena/brows er/view/PRJEB 35111) and Trypanosoma sequences are available on Genbank (Accessions MT572485-MT572490).