First Molecular Identification of Trypanosomes and Absence of Babesia sp. DNA in Faeces of Non-Human Primates in the Ecuadorian Amazon

Trypanosomes are a group of pathogens distributed in the continents of Africa, America, Asia and Europe, and they affect all vertebrates including the neotropical primate group. Information about the trypanosome’s diversity, phylogeny, ecology and pathology in non-human primates (NHPs) from the neotropical region is scarce. The objective of the study was to identify Trypanosoma and Babesia molecularly in NHPs under the phylogenetic species concept. We extracted DNA from a total of 76 faecal samples collected between 2019 and 2021, from a total of 11 non-human primate species of which 46 are from captive NHPs and 30 are free-living NHPs in the Western Amazon region of Ecuador. We did not detect DNA of Babesia sp. by polymerase chain reaction test in any of the faecal samples. However, the nested-PCR-based method revealed Trypanosoma parasites by ITS gene amplification in two faecal samples; one for the species Leontocebus lagonotus (from the captive population) and a second one for Cebus albifrons (from the free-ranging population). Maximum parsimony and likelihood methods with the Kimura2+G+I model inferred the evolutionary history of the two records, which showed an evolutionary relationship with the genus Trypanosoma. Two sequences are monophyletic with Trypanosoma. However, the number of sequences available in GenBank for their species identification is limited. The two samples present different molecular identifications and evolutionary origins in the tree topology. We are most likely referring to two different species, and two different localities of infection. We suggest that health management protocols should be implemented to prevent the transmission of blood-borne pathogens such as Trypanosoma sp. among captive populations. In addition, these protocols also protect the personnel of wildlife rehabilitation centers working in close proximity to NHPs and vice versa.

In Ecuador, information regarding blood pathogens in NHPs is scarce. Therefore, this study aimed for the first time to detect Trypanosoma and Babesia in captive and free-ranging NHPs from the Ecuadorian Amazon through molecular techniques (PCR and sequencing).

Sampling Location
This study was performed in Puyo (1 •

Sample Collection and Ethics Statement
We collected a total of 76 fecal samples of 11 species of NHPs (Table 1) between 2019 and 2021. The sample collection and ethical procedures were approved by the local authorities, the Ministerio del Ambiente, Agua y Transición Ecológica, MAATE (No. MAE-DNB-CM-2015-0028-M-002). Individuals were followed daily from 08:00 h to 18:00 h to avoid multiple sampling. In addition, all animals were individually identified to avoid confusion between individuals and to facilitate species, sex and age association [92]. Finally, we collected the faecal samples immediately after defecation to avoid a possible contamination from the environment and were taken at least 24 h without disturbing the animals. In primatology, when birth dates are unknown, age as well as sex, is generally assigned in categories based on physical characteristics including body size, dentition and gland development in species where these are evident, and behavioral characteristics [93][94][95]. All samples were processed at the International Centre of Zoonoses at the Central University of Ecuador and examined at the Biotechnology Animal Lab at the Universidad de las Fuerzas Armadas ESPE.

Storage and DNA Isolation Protocol
For the molecular evaluation, samples were stored in 50 mL Falcon tubes in 99% alcohol at −20 • C to prevent the degradation of DNA. In addition, 600 µL of faeces suspension (1:3; 1 part of faecal sample and 3 parts of ethanol 96-100%) was centrifuged for 2 min at 239 g and the pellet was washed with 1 mL of PBS Buffer (Oxoid, Hampshire, England). This solution (pellet + PBS) was centrifuged for 5 min and the supernatant was discarded. This washing step was repeated three times. Next, the pellet was resuspended in 600 µL of 2% PVPP (polyvinylpolypyrolidone-Sigma), and frozen overnight at −20 • C to facilitate the capture of phenols in the sample. DNA extraction was performed twice on different days using the QIAamp Stool FAST Mini Kit (Qiagen GmbH, Hilden, Germany) following the manufacturer's instructions. To prevent cross-contamination, sample preparation, DNA extraction, and the polymerase chain reaction (PCR) were performed in completely different and separate rooms.

Molecular Amplification and Sequencing of Trypanosoma sp.
The molecular identification was performed with two PCR assays: (i) A CatL-PCR, according to reaction conditions by Cortez et al. [70] (Table 2), with adaptations in the reaction mixture, that consisted of a final volume of 25 µL with 1× of Buffer, 1 µM of each primer DTO154 and DTO155 (Table 3), 1.5 mM of MgCl 2 , 0.2 mM of dDNT Mix, 1.25 U of Taq Platinum Polymerase (Invitrogen) and 2 µL of DNA. (ii) A nested ITS-PCR as described by [96,97], the reaction mixture consisted of 1× of Buffer, 1 µM of each primer (ITS1 and ITS2 in the first reaction, ITS3 and ITS4 in the second reaction) (Table 3), 2.5 mM of MgCl 2 , 0.2 mM of dDNT Mix, 1.25 U of Tap Platinum Polymerase (Invitrogen) and 2 µL of DNA in the first reaction and 1uL of first reaction PCR product in the second reaction; the amplification consisted of an initial denaturation of 95 • C for 7 min; 35 cycles of 95 • C for 1 min, 59 • C for 1 min, 72 • C for 2 min in the first reaction and 1.5 min in the second reaction; and a final extension step at 72 • C for 10 min ( Table 4). The final PCR products of the CatL-PCR and ITS-PCR were observed using the electrophoresis of an agarose gel under UV light. ITS-PCR amplicons were cut, extracted using the Wizard ® SV Gel and PCR Clean-Up System (Promega) and sequenced (Sanger sequencing) by Macrogen (South Korea). Every PCR reaction contained a negative (nuclease-free water) and a positive control. A positive control for CatL-PCR was a positive DNA sample of Trypanosoma vivax [98] and for ITS-PCR a positive DNA sample of Trypanosoma theileri, available in the Laboratorio de Biotecnología Animal of the Universidad de las Fuerzas Armadas ESPE. Two sequences belonging to the 5.8S and ITS-2 were recovered from Sanger sequencing Macrogen Korea. Step Temperature Time

Number of Cycles
Step 1 Pre-denaturation 94 • C 5 min 1 cycle Step 2  Table 4. Three steps nested ITS-PCR cycles, temperature, and time for Trypanosoma sp.
Step Temperature Time

Number of Cycles
Step 1 Pre-denaturation 95 • C 7 min 1 cycle Step 2 The molecular identification by PCR was performed with the primers designed by Olmeda et al. [99] and using the reaction condition described by Medina Naranjo et al. [100], which consisted of: 1× of Buffer, 0.25 µM of each primer Piro A and Piro B (Table 3) Table 5). The PCR products were observed using the electrophoresis of an agarose gel under UV light. Every PCR reaction contained a negative and a positive control. A positive control for Babesia sp. was a DNA sample obtained from the study performed by Chávez-Larrea et al. [101]. Table 5. Three steps PCR cycles, temperature, and time for Babesia sp.
Step Temperature Time

Number of Cycles
Step 1 Pre-denaturation 94 • C 5 min 1 cycle Step 2

Molecular Analysis: Sequence Assembled, Alignments and Phylogenetic Analyses
Sequences were uploaded to GenBank under the accession number OP683488.1 for Trypanosoma sp. detected in Leontocebus lagonotus and OP683532.1 for Trypanosoma sp. detected in Cebus albifrons. Our two 5.8S-ITS-2 sequences of 400 bp length were contig assembly and consensus sequences of ITS-2 were performed and edited using Assembler by MacVector software 18.2.5 [9], then the sequence identity was confirmed by BLAST in NCBI resources. The two sequences were first aligned with the unique complete sequence 18S-ITS1-5.8S-ITS2-28S available in GenBank that included ITS2 (T. minansense AB362411.1 by Sato et al. [102], recovered from new-world NHPs from South American tamarins) to corroborate the portion of rDNA and matching or sequences (from 2636 bp to 3019 bp). The 5.8S-ITS-2 sequences deposited in GenBank NCBI from other species of Trypanosoma were included as sister groups and Leishmania was selected as the outgroup sensu [103] to get a wide geographic diversity and taxonomic representative and to test the phylogenetic species monophyly.
A phylogenetic analysis was performed using a total of 23 ITS-2 sequences from 13 species of Trypanosoma that were retrieved from GenBank and included two species of Leishmania as outgroup to corroborate the Blast identity close to Trypanosomatidae and to search the evolutionary relationships with other trypanosomes ( Table 6).
The DNA sequences were aligned using MacVector 18.2.5 by the ClustalW algorithm with high gap creation and extension penalties by 30.0 and 10.0, respectively, searching for a strong positional homology.
The evolutionary history was inferred by using the maximum parsimony and likelihood methods with the Kimura2+G+I model. Maximum Parsimony analyses were implemented in PAUP 4.0a (169 build) [104] using the heuristic search option with a Tree Bisection Reconnection branch-swapping algorithm with at-random stepwise addition of 10 replicates for each search and 100-1000 replications per analysis. Gaps were treated both as missing data. The characters were treated as unordered, and equally weighted, after the characters were weighted by consistency index. The robustness of the trees was estimated using parsimony bootstrap with 1000 pseudoreplicates after excluding uninformative characters [105]. We also performed a Maximum Likelihood (ML, and substitution model estimated on MEGAX). Table 6. Sequences obtained from GenBank to elaborate the cladograms.

Species/Sequences
GenBank ID

Results
We have performed a PCR and a Nested PCR to detect the gene Trypanosoma from faecal samples of 11 species of NHPs. The PCR with the Catepsine L-Like gene failed to amplify DNA from Trypanosoma sp., although this gene can be easily amplified in other samples from wild mammals [106]. On the other hand, the Nested PCR of 35 cycles successfully amplified the ITS1 gene.
We did not observe positive samples for Babesia sp. for any of the samples. From the 458 Trypanosoma sequences in GenBank that partially or completely included ITS-2 belonging to 16 species, we aligned the two ITS sequences amplified from the faecal samples with 21 ITS sequences from 13 species (Table 3) (three species from Russia were not included) to elaborate the cladogram.
The cladogram showed that our two sequences belong to the trypanosomes genus. Our results revealed two unexpected novel sequences. The topology of the cladogram (Figure 2A Clade B shows the sequence of Cebus albifrons PM020 from Ecuador, internally and closely related to T. congolense and basal to T. vivax + [(Trypanosmatidae sp (T. godfreyi + T. simiae)], showing its close relationship with these species. Likewise, there is no ITS-2 sequence in GenBank that shows monophyly with our sequence for a specific identification.
The topologies using maximum parsimony (MP) and maximum likelihood (ML) under the Kimura2+G+I model showed identical relationships ( Figure 2B), the two Leishmania species as outgroup allow corroborating the monophyly of Trypanosoma (100% bootstrap), as well as our sequences showing their evolutionary relationship within the genus. sequence in GenBank that shows monophyly with our sequence for a specific identification.
The topologies using maximum parsimony (MP) and maximum likelihood (ML) under the Kimura2+G+I model showed identical relationships ( Figure 2B), the two Leishmania species as outgroup allow corroborating the monophyly of Trypanosoma (100% bootstrap), as well as our sequences showing their evolutionary relationship within the genus.

Discussion
The present study is the first at the national level and one of the few at the regional level to identify two species of Trypanosoma sp using non-invasive techniques. Although the ITS region was successfully amplified in wild gorillas (Gorilla gorilla gorilla) and chimpanzees (Pan troglodytes troglodytes) for trypanosomes, this is the first study in the neotropical region to use the ITS region to amplify trypanosomes in faecal samples. We identified the first record of Trypanosoma for the NHP species Leontocebus lagonotus and the

Discussion
The present study is the first at the national level and one of the few at the regional level to identify two species of Trypanosoma sp. using non-invasive techniques. Although the ITS region was successfully amplified in wild gorillas (Gorilla gorilla gorilla) and chimpanzees (Pan troglodytes troglodytes) for trypanosomes, this is the first study in the neotropical region to use the ITS region to amplify trypanosomes in faecal samples. We identified the first record of Trypanosoma for the NHP species Leontocebus lagonotus and the first report for Ecuador of Trypanosoma in Cebus albifrons. We detected 10.53% of positive samples, whereas only 2.63% yielded a positive sequence. This prevalence is lower than other studies [107,108]. In Aysanoa et al. [22], they found a lower prevalence in captive NHP individuals than in wild individuals. Captive animals may be subject to liberation projects, and they can introduce new trypanosomatids to liberation sites. Triatomines were found in a Brazilian zoo, infesting neotropical NHPs. This indicates that the same pattern could be possible in Ecuadorian wildlife rehabilitation centers where vegetation could facilitate the presence of trypanosomes vectors. Common vectors of trypanosomes like triatomine bugs (Panstrongylus geniculatus, Triatoma dimidiata, Rhodnius pictipes and Rhodnius robustus) can be found in the Ecuadorian Amazon [17,109] and the proximity of the forests to the centers would facilitate the maintenance of the forest cycle.
Previous studies have suggested that trypanosomes tend to have harmful effects on the health of infected hosts [110][111][112][113][114]. However, information on the effect of triatomines in NHPs is scarce. The individuals who tested positive had no obvious symptoms that would allow us to make a statement about their health condition, noting that trypanosome records have been made in healthy individuals as well as in sick individuals.
After phylogenetic reconstruction, we identified two large groups: the first, in which we found the sample of Leontocebus lagonotus within the same cladogram as the species of T. brucei, T. equiperdum, T. evansi, T. cruzi, T. minasense and T. theileri. In the second cladogram, we found the sample of Cebus albifrons together with the species of: T. simiae, T. godfreyi, T. vivax and T. congolense. This distribution coincides with several authors [102]. The two samples show different molecular identifications and evolutionary origins, certainly two species, and two localities of infection.
The use of molecular tools for the detection of Trypanosoma spp. is crucial because of the unreliability of detection methods based on the observation of their morphology [115,116]. The two sequences are shown to be in monophyly with Trypanosoma; however, there are not enough sequences available in GenBank for their specific identification.
As mentioned before, the gene from the Cathepsin L-like protein failed to amplify in the faecal samples. However, past studies diagnosed T. rangeli, T. cruzi and T. theileri with this gene [71,106,117,118]. This protein is a lysosomal cysteine proteinase. The Cathepsin L-like protein is found in several stages of cell multiplication and differentiation as well as cell metabolism and virulence (host cell invasion, immune evasion) in protozoan parasites such as trypanosomes [119,120]. However, according to Cortez et al. [70], this gene has a different number of copies depending on the trypanosome species and therefore is speciesspecific, and because we were surveying trypanosomes in general, the protocol failed to amplify for all trypanosome species and specifically for neotropical NHPs' trypanosomes, given what was observed with the amplification of the ITS gene.
Based on the findings, the ITS gene is a useful molecular marker to detect trypanosomes; however, for further studies, it is suggested to amplify with 18S-ITS-1 (higher availability); this combination would support us in defining in more specific detail the molecular characterization of these two records. Unfortunately, only T. simiae, T. rangeli, T. cruzi and T. minasense (included) of the NHPs-associated Trypanosoma have ITS2 sequences available in GenBank from the list in Appendix A.
We did not record the presence of Babesia sp., a protozoan pathogen with a worldwide distribution restricted to tropical and subtropical areas [101]. Non-human primates are a group of mammals that have generated strategies to prevent pathogens [121][122][123]. One of these strategies is grooming. Grooming is a behaviour that directly supports health-related aspects of different primate species, including the removal of ectopathogens such as leeches in Macaca fuscata [124] and ticks [125,126]. For Papio cynocephalus (Africa), it was recorded that the amount of grooming received, sex, age and hierarchical level affected the tick load of an individual. However, the primary function of grooming contributes to social aspects in different old=world primate species [127], whereas, in new-world primates, it is suggested that the main function of grooming is hygienic [128]. For this reason, this type of grooming can explain the absence of Babesia sp. in our study. It is important to conduct long-term studies that allow us to relate the presence of ticks to the prevalence/absence of tick-borne diseases in non-human primates.

Conclusions
This is the first study to amplify trypanosomes in Ecuadorian NHP species. Even if the prevalence was low, we suggest the implementation of health management protocols to avoid the transmission of blood-borne pathogens such as Trypanosoma sp. among captive populations. In addition, these protocols protect the personnel of wildlife rehabilitation centers working in close proximity to NHPs and vice versa. Socioecological aspects are of utmost importance to understand pathogen-vector-host relationships in different species of NHPs. In Ecuador, research activities should be focused on blood pathogens to fill the gap of information and to implement surveillance programs with regular and effective monitoring protocols adapted to NHPs. We suggest to increase the monitoring of free-ranging groups across Ecuador to clarify the role of NHPs as reservoir hosts of novel trypanosomes.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest:
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.