Intestinal parasites infecting free-ranging primates in Colombia: Morphological and molecular evidence

Abstract

Intestinal protozoan and metazoan parasites of public health concern have been found infecting non-human primates (NHPs). Since zoonotic parasite transmission can be linked to ecosystem transformation due to close contact between human and NHPs, this study aimed to morphologically identify intestinal parasites infecting NHPs living in seven forest fragments in Colombia and to molecularly characterise selected parasite taxa of zoonotic interest (Giardia sp. and Ascaris sp.). Building on and updating previous parasite surveys on free-ranging platyrrhines, 212 faecal samples were collected from wild Alouatta seniculus, Ateles hybridus, Aotus griseimembra, Cebus versicolor, Saimiri cassiquiarensis, and Sapajus apella. Flotation and faecal smears were performed in order to identify parasites based on morphological characteristics. Samples microscopically classified as positive for Giardia sp. and Ascaris sp. were then processed for molecular characterisation. Amplification of the triosephosphate isomerase (TPI) and the 18S genes was done for Giardia sp., while for Ascaris sp. a PCR-RPLF of the entire ITS region was carried out. About 96% of the samples were positive for intestinal parasites, including: protozoans (Blastocystis sp., Balantidium sp., Dientamoeba fragilis-like, Entamoeba sp., Giardia sp., Eimeria sp.), nematodes (Ascaris sp., strongyliform larvae, Trypanoxyuris sp., Ancylostomatidae), trematodes (Controrchis sp., Trematoda), cestodes (Hymenolepis sp., Cestoda), and acanthocephalans. By microscopy, the prevalence of Giardia sp. and Ascaris sp. was 16.5% and 0.9%, respectively. Later on, Giardia duodenalis (Assemblages A and B) and Ascaris lumbricoides were identified through molecular techniques. This study provides new information of intestinal parasites infecting free-ranging NHPs exposed to anthropogenic disturbance. The finding of parasites with zoonotic potential suggests epidemiological implications in NHP conservation and human health, at the human-NHP interface.

Graphical abstract

Highlights

• •Fifteen parasite taxa were recorded: six protozoans, four nematodes, two trematodes, two cestodes, and acanthocephalans.
• •Giardia duodenalis (assemblages A and B) was identified through amplification of the triosephosphate isomerase (TPI) and the 18S genes.
• •Ascaris lumbricoides was identified through PCR-RPLF of the entire ITS region.
• •Parasite prevalence was not related to primate species nor study site, unlike parasite richness which was related to both.

1. Introduction

In Latin America, 36% of non-human primate (NHP) taxa are considered to be endangered and almost two thirds of them have declining populations (Estrada et al., 2017). Colombia is home to 38 NHP species and 14 subspecies (Link et al., 2018). Of these, three are considered Critically Endangered, five Endangered, and fifteen Vulnerable according to the Red List of the International Union for Conservation of Nature (IUCN, 2022). Colombia is a mega-diverse country with high anthropogenic transformation of ecosystems (Correa Ayram et al., 2020).

Anthropogenic habitat modification can impact disease dynamics via changes in resource availability and distribution. For instance, food provisioning has been associated with increased host densities, endoparasite infections and inter-individual contact rates. Furthermore, humans also introduce pathogens into novel areas often via domestic animals (Hammond et al., 2020). From a public health perspective, parasitological surveys are pertinent both for NHP and human welfare, as ecosystem transformation (e.g. Deforestation, habitat fragmentation), and increased contact between humans and NHPs could facilitate the chances of zoonotic parasite transmission.

Diverse intestinal protozoan and metazoan parasites have been found infecting NHPs (Li et al., 2015; Solórzano-García and Pérez-Ponce de León, 2018). Some of those parasites are of public health concern, such as Giardia, which has global importance infecting a wide range of hosts and causing ≈28.2 million cases of diarrhoea each year (Ryan et al., 2019), as well as the nematode Ascaris lumbricoides included in the main species causing soil-transmitted helminth infections recognized as neglected tropical diseases with a global burden of 1.5 billion people infected worldwide (World Health Organization, 2022).

In Colombia, parasite studies involving NHPs include the following taxa: Alouatta seniculus, Aotus griseimembra, Aotus lemurinus, Aotus nancymaae, Aotus trivirgatus, Aotus vociferans, Ateles hybridus, Ateles geoffroyi, Callicebus cupreus, Cebus albifrons, Cebus versicolor, Lagothrix lagotricha, Saguinus leucopus, Saguinus oedipus, Saimiri sciureus, and Sapajus apella (Rondón et al., 2021; Solórzano-García and Pérez-Ponce de León, 2018). However, surveys are still scarce and mostly conducted on captive NHPs or under laboratory conditions, while studies on free-ranging NPHs are less frequent, and in general, mostly based on parasite identification through morphology and only a few using molecular markers. Studies using molecular techniques have identified the protozoans Cryptosporidium, Blastocystis, Giardia, Plasmodium, and Leishmania (Castillo-Castañeda et al., 2022; Cruz-Saavedra et al., 2023; Rondón et al., 2019, 2023b), and few metazoan species such as Trypanoxyuris spp. and Cuterebra baeri (Rondón et al., 2023a; Solórzano-García et al., 2020).

Morphological identification has unique value and is the gold standard for parasite diagnosis (Bradbury et al., 2022), however, some parasite species and/or genetic variants could not be determined based on parasite morphology thus not allowing to assess their zoonotic potential. Therefore, molecular approaches are necessary as a complement to microscopy in order to better understand the epidemiology and to infer the zoonotic potential of parasites. In addition, it is necessary to conduct parasitological sampling in different geographical areas aiming to understand transmission dynamics in different ecological scenarios.

To build on and update previous parasite surveys in free-ranging platyrrhines, this study aimed to morphologically identify intestinal parasites infecting NHPs living in seven forest fragments in Colombia, to estimate their prevalence and species richness, and to molecularly characterise selected parasite taxa of zoonotic interest (e.g., Giardia sp. and Ascaris sp.).

2. Material and methods

2.1. Sampling

Fieldwork was carried out between December 2019 and February 2022, in seven localities (San Juan, Cumaral, Cabuyaro, Guacavía, Villavicencio, Maní, Yopal) in Santander, Meta and Casanare Departments, in Colombia. Free-ranging primates were followed from dawn to dusk, and one faecal sample per individual was collected from the soil immediately after defecation. For each sample, one aliquot was stored in 10% formalin solution, and another aliquot in 96% ethanol solution. Overall, 212 samples were collected from six primate species, including Alouatta seniculus, Aotus griseimembra, Ateles hybridus, Cebus versicolor, Sapajus apella and Saimiri cassiquiarensis (Table 1 and Fig. 1).

Table 1.

Study sites information.

Study site	Coordinates	Department	Primate species	N samples
San Juan	06°43′ N 74°09′ W	Santander	Alouatta seniculus	28
			Cebus versicolor	20
			Ateles hybridus	13
			Aotus griseimembra	5
Cumaral	04°17′ N 73°24′ W	Meta	Saimiri cassiquiarensis	42
Villavicencio	04°06′ N 73°38′ W	Meta	Saimiri cassiquiarensis	24
Guacavía	04°17′ N 73°30′ W	Meta	Saimiri cassiquiarensis	30
Cabuyaro	04°17′ N 73°02′ W	Meta	Sapajus apella	3
Maní	04°48′ N 72°19′ W	Casanare	Alouatta seniculus	18
Yopal	05°16′ N 72°22′ W	Casanare	Sapajus apella	18
			Alouatta seniculus	11

Fig. 1.

Study sites and number of samples collected per primate species and study site.

2.2. Morphological analyses

Faecal samples stored in 10% formalin solution were used to perform smears with 1% iodine solution and 0.85% saline solution (Botero, D & Restrepo, 2012). For each sample, two microscope slides were mounted and systematically examined under a microscope using magnifications at 100x, 400x and 1000x. Additionally, flotation with a salt-sugar solution (SG: 1.28) was performed (Rondón et al., 2023b). Morphological identification of protozoan and helminth parasites was done after direct faecal smears and flotation. Photos of parasites (larvae, cysts, oocysts) and measures using the microscope camera lucida were taken for posterior identification using taxonomic keys (Genchi et al., 2010; World Health Organization, 2019).

2.3. Molecular analyses

For faecal samples microscopically classified as positive for Giardia sp. and Ascaris sp. the corresponding aliquots stored in 96% ethanol solution were individually subjected to DNA extraction, using the Isolate II Fecal DNA Kit (Meridian Bioscience) according to the manufacturer's protocol.

For Giardia sp., two PCRs were carried out using the primers RH11-RH4 to amplify a 292 bp fragment of the 18S ribosomal subunit gene, and the primers AL3543-AL3246 and AL3544-AL3545 to amplify a 530 bp fragment of the triosephosphate isomerase (TPI) gene. PCR conditions for the 18 ribosomal subunit gene were 96 °C for 2 min followed by 35 cycles of 96 °C for 20 s, 59 °C for 20 s, and 72 °C for 30 s, and 1 cycle of 72 °C for 7 min (Hopkins et al., 1997). For the TPI gene, a primary PCR with the primers AL3543-AL3246 was conducted with an initial hot start (94 °C for 5 min), followed by 35 cycles (94 °C for 45 s, 50 °C for 45 s, and 72 °C for 60 s), and a final extension (72 °C for 10 min). A secondary PCR with the primers AL3544-AL3545 was carried out with the same PCR conditions to the primary PCR (Sulaiman et al., 2003). All PCR products were visualized on a 1.5% agarose gel stained with SYBR Safe and positive samples were purified using Sure Clean Plus (Bioline, UK) and shipped to an external company for sequencing (Eurofins Genomics). Forward and reverse sequences were manually edited using Trace implemented in MEGA7 (Kumar et al., 2016) for quality check and inference of consensus sequences and thereafter were used as input for BLAST search and assemblage assignment using the PubMLST.org website and reference datasets for each genus, according to the most recent available literature (Supplementary Tables 1 and 2). For Giardia sp. obtained sequences, alignments were carried out per each partial region of the studied genes, using ClustalW implemented in MEGA7. A first comparison aimed to confirm the assemblage identity and to explore the phylogenetic relationships was performed, including the present data and the reference sequences of all available Giardia assemblages (Supplementary table 2). Assemblage H was not included in the analyses since homologous sequences are missing in public repositories. Verified sequences here obtained were submitted to GenBank and the accession numbers are listed in Supplementary Tables 1 and 2

Using ModelTest implemented in MEGA7 the best evolutionary models were obtained. The Maximum Likelihood (ML) method was used to infer phylogenies and confirm the relationships among Giardia samples with the other assemblages. Statistical support at nodes were obtained using the bootstrap method with 1000 pseudo replications.

For Ascaris positive samples, a conventional PCR was carried out in order to amplify the entire ITS nuclear ribosomal region, using the forward primer NC5 and the reverse primer NC2. PCR conditions were 10 min at 95 °C, followed by 30 cycles of 30 s at 95 °C, 40 s at 52 °C, and 75 s at 72 °C, and a final elongation step of 7 min at 72 °C (Cavallero et al., 2013). PCR products were visualized on a 1% agarose gel stained with SYBR Safe. To differentiate Ascaris lumbricoides and Ascaris suum, positive amplicons were digested with the HaeIII restriction endonuclease (Nejsum et al., 2005), followed by an electrophoresis in a 2% agarose gel stained with SYBR Safe.

2.4. Data analyses

The Quantitative Parasitology on the Web Software (QPweb) version 1.0.15 (Reiczigel et al., 2019) was used to calculate the prevalence of parasites per study site and primate species, with 95% confidence intervals. Likewise, parasite richness was estimated as the number of taxa recovered from the host's faecal samples.

For Blastocystis sp., Giardia sp., strongyliform larvae, and Entamoeba sp. additional significance tests based on Fisher's were performed using the Quantitative Parasitology Software to compare prevalence values among sites (same primate species from different study sites) and species (different primate species from the same study site).

The RStudio integrated development environment for the R Software (RStudio Team, 2020; PBC, Boston MA) was used to test normal distribution and then to perform a two-way analysis of variance (ANOVA) in order to evaluate the effect of primate species and study site on the prevalence and richness of parasites, using a dataset that included the primate species for which more than 30 samples were collected (A. seniculus and S. cassiquiarensis).

3. Results

3.1. Morphological analyses

A very high prevalence of infection was found, with 96% (N = 203) of the samples positive for intestinal parasites. Co-infections with two to five parasite taxa were recorded in 139 samples. Overall, 15 parasite taxa were found, including protozoans (Blastocystis sp., Balantidium sp., Dientamoeba fragilis-like, Entamoeba sp., Giardia sp., Eimeria sp.), nematodes (Ascaris sp., strongyliform larvae, Trypanoxyuris sp., Ancylostomatidae), trematodes (Controrchis sp., Trematoda), cestodes (Hymenolepis sp., Cestoda), and acanthocephalans (Fig. 2).

Fig. 2.

Representative images from the parasite taxa observed by microscopy. A. Entamoeba sp. (30 μm), B. Giardia sp. (12 × 8 μm), C. Blastocystis sp. (8 μm), D. Eimeria sp., E. Trypanoxyuris sp. (45 × 20 μm), F. Balantidium sp. (40 × 45 μm), G. Strongyliform larvae (40x), H. Controrchis sp. (35 × 20 μm), I. Ascaris sp. (40 × 75 μm), J. Acanthocephala (40 × 70 μm) K. Hymenolepis sp. (50 μm), L. Ancylostomatidae (36 × 60 μm).

3.2. Molecular analyses

Regarding Giardia sp., eight high-quality sequences for the 18S gene, including one from A. seniculus from Maní, two of S. apella and four of A. seniculus from Yopal, and one of A. seniculus from San Juan were obtained, while for the TPI gene, we obtained six high-quality sequences, including one of A. seniculus from San Juan, and one of S. apella and four of A. seniculus from Yopal. For five faecal samples, sequences for both 18S and TPI genes were obtained. According to the best match in BLAST all sequences were identified as Giardia duodenalis, with 95.2–100% identity. Assemblage assignment was done using the PubMLST website and a 297bp alignment with 19 variable positions. Results obtained for the partial 18S dataset by ML phylogenetic inferences with the T92 model reported the presence of assemblages A and B in the material here analysed from NHPs from Colombia (Fig. 3a). Results for the TPI dataset (Fig. 3b) confirmed the main assemblage affiliation of the sequences, which clustered in the branch of assemblage B4 and A1-A2.

Fig. 3.

Best ML consensus tree of a. Partial 18S Giardia duodenalis reference assemblages and samples here analysed collected from NHPs in Colombia (Assemblage E was removed for length diversity of input sequences), b. Partial TPI Giardia duodenalis reference assemblages and samples here analysed collected from NHPs in Colombia.

For the two faecal samples from S. cassiquiarensis from Cumaral microscopically classified as positive for Ascaris sp., a PCR product of around 1000 bp was obtained. After the digestion with the HaeIII restriction enzyme, a banding pattern belonging to the “lumbricoides” genotype displaying two bands of approximately 610 bp and 370 bp.

3.3. Data analyses

The prevalence of each parasite taxa according to primate species and study sites is shown in Table 2A, Table 2B, Table 2CA, 2B, and 2C, while parasite richness is indicated in Table 3.

Table 2A.

Parasite prevalence (%) in faecal samples per NHP species, for the two study sites at Casanare department. Confidence limits for the prevalence are indicated between square brackets.

Parasite taxa	Maní	Yopal
	Alouatta seniculus (n = 18)	Alouatta seniculus (n = 11)	Sapajus apella (n = 18)
Blastocystis sp.∗	8 (44.4%) [21.5%–69.2%]	5 (50.0%) [18.7%–81.3%]	5 (27.8%) [9.7%–53.5%]
Dientamoeba fragilis∗∗	12 (66.7%) [41.0%–86.7%]	2 (18.2%) [2.3%–51.8%]
Entamoeba sp.		3 (27.3%) [6.0%–61.0%]
Giardia sp.	4 (22.2%) [6.4%–47.6%]	6 (54.5%) [23.4%–83.3%]	6 (33.3%) [13.3%–59.0%]
Strongyliform larvae∗∗∗		5 (45.5%) [16.7%–76.6%]	18 (100%) [81.5%–100%]
Trypanoxyuris sp.	2 (11.1%) [1.4%–34.7%]	2 (18.2%) [2.3%–51.8%]
Hymenolepis sp.			2 (11.1%) [1.4%–34.7%]

^∗Prevalence and molecular analyses on Blastocystis sp. from this sampling have been already published (Rondón et al., 2023b).

^∗∗Dientamoeba fragilis is suspected, confirmation is needed.

^∗∗∗Based on morphology it was not possible to identify the genus/species.

Table 2B.

Parasite prevalence (%) in faecal samples per NHP species, at Santander department. Confidence limits for the prevalence are indicated between square brackets.

Parasite taxa	San Juan
	Alouatta seniculus (n = 28)	Aotus griseimembra (n = 5)	Ateles hybridus (n = 13)	Cebus versicolor (n = 20)
Blastocystis sp.∗	21 (75.0%) [55.1%–89.3%]		7 (53.8%) [25.1%–80.8%]	6 (30.0%) [11.9%–54.3%]
Balantidium sp.	2 (7.1%) [0.9%–23.5%]		10 (76.9%) [46.2%–95.0%]
Dientamoeba fragilis∗∗	11 (39.3) [21.5%–59.4%]		1 (7.7%) [0.2%–36.0%]
Entamoeba sp.	5 (17.9%) [6.1%–36.9%]	1 (20.0%) [0.5%–71.6%]	1 (7.7%) [0.2%–36.0%]	3 (15.0%) [3.2%–37.9%]
Giardia sp.	5 (17.9%) [6.1%–36.9%]	1 (20.0%) [0.5%–71.6%]	1 (7.7%) [0.2%–36.0%]	2 (10.0%) [1.2%–31.7%]
Eimeria sp.	1 (3.6%) [0.1%–18.3%]	1 (20.0%) [0.5%–71.6%]		1 (5.0%) [0.1%–24.9%]
Ancylostomatidae				1 (5.0%) [0.1%–24.9%]
Strongyliform larvae∗∗∗	4 (14.3%) [4.0%–32.7%]	2 (40.0%) [5.3%–85.3%]	7 (53.8%) [25.1%–80.8%]	19 (95.0%) [75.1%–99.9%]
Trypanoxyuris sp.	2 (7.1%) [0.9%–23.5%]
Controrchis sp.				10 (50.0%) [27.2%–72.8%]
Trematoda				3 (15.0%) [3.2%–37.9%]
Acanthocephala				4 (20.0%) [5.7%–43.7%]

^∗Prevalence and molecular analyses on Blastocystis sp. from this sampling have been already published (Rondón et al., 2023b).

^∗∗Dientamoeba fragilis is suspected, confirmation is needed.

^∗∗∗Based on morphology it was not possible to identify the genus/species.

Table 2C.

Parasite prevalence (%) in faecal samples per NHP species, for the four study sites at Meta department. Confidence limits for the prevalence are indicated between square brackets.

Parasite taxa	Cabuyaro	Cumaral	Guacavía	Villavicencio
	Sapajus apella (n = 3)	Saimiri cassiquiarensis (n = 42)	Saimiri cassiquiarensis (n = 30)	Saimiri cassiquiarensis (n = 24)
Blastocystis sp.∗	1 (33.3%) [0.8%–90.6%]	11 (26.2%) [13.9%–42.0%]
Dientamoeba fragilis∗∗		10 (23.8%) [12.1%–39.5%]	1 (3.3%) [0.1%–17.2%]
Entamoeba sp.		4 (9.5%) [2.7%–22.6%]	3 (10.0%) [2.1%–26.5%]	2 (8.3%) [1.0%–27.0%]
Giardia sp.		3 (7.1%) [1.5%–19.5%]	3 (10.0%) [2.1%–26.5%]	4 (16.7%) [4.7%–37.4%]
Eimeria sp.			1 (3.3%) [0.1%–17.2%]
Ancylostomatidae	1 (33.3%) [0.8%–90.6%]
Ascaris sp.		2 (4.8%) [0.6%–16.2%]
Strongyliform larvae∗∗∗	2 (66.7%) [9.4%–99.2%]	38 (90.5) [77.4%–97.3%]	25 (83.3%) [65.3%–94.4%]	23 (95.8%) [78.9%–99.9%]
Trypanoxyuris sp.		1 (2.4%) [0.1%–12.6%]	3 (10.0%) [2.1%–26.5%]	1 (4.2%) [0.1%–21.1%]
Controrchis sp.	1 (33.3%) [0.8%–90.6%]	4 (9.5%) [2.7%–22.6%]	1 (3.3%) [0.1%–17.2%]
Trematoda		4 (9.5%) [2.7%–22.6%]		1 (4.2%) [0.1%–21.1%]
Hymenolepis sp.		1 (2.4%) [0.1%–12.6%]	1 (3.3%) [0.1%–17.2%]
Cestoda		1 (2.4%) [0.1%–12.6%]	2 (6.7%) [0.8%–22.1%]
Acanthocephala		29 (69.0%) [52.9%–82.4%]	19 (63.3%) [43.9%–80.1%]	9 (37.5%) [18.8%–59.4%]

^∗Prevalence and molecular analyses on Blastocystis sp. from this sampling have been already published (Rondón et al., 2023b).

^∗∗Dientamoeba fragilis is suspected, confirmation is needed.

^∗∗∗Based on morphology it was not possible to identify the genus/species.

Table 3.

Parasite richness per NHP species and study sites.

Study site	Primate species	Parasite richness (N parasite taxa)
San Juan	Alouatta seniculus	8
	Cebus versicolor	9
	Ateles hybridus	6
	Aotus griseimembra	4
Cumaral	Saimiri cassiquiarensis	12
Villavicencio	Saimiri cassiquiarensis	6
Guacavía	Saimiri cassiquiarensis	10
Cabuyaro	Sapajus apella	4
Maní	Alouatta seniculus	4
Yopal	Sapajus apella	4
	Alouatta seniculus	6

Parasite prevalence values obtained for the same primate species from different study sites and from different primate species from the same study site revealed significant differences on Blastocystis sp. prevalence for S. cassiquiarensis from Cumaral, Guacavía and Villavicencio (p = 0.0002), being present only in Cumaral; and for A. hybridus, C. versicolor and A. seniculus from San Juan (p = 0.001), being absent only in A. griseimembra. Similarly, Entamoeba sp. prevalence values were significantly different in A. seniculus from San Juan, Maní and Yopal (p = 0.05), being more prevalent in Yopal and absent in Maní. Prevalence values for strongyliform larvae were significantly different in A. seniculus from San Juan, Maní and Yopal (p = 0.003), with a higher prevalence in Yopal while absent in Maní; and in different primate species (A. hybridus, A. griseimembra, C. versicolor and A. seniculus) from San Juan (p < 0.001), and (A. seniculus and S. apella) from Yopal (p = 0.001). Comparisons performed for Giardia sp. prevalence were not significant.

Anova and Tukey's tests for the dataset of A. seniculus and S. cassiquiarensis showed that parasite prevalence was not related to primate species (Anova: p-value = 0.718, F-value = 0.131, Df = 1), nor to study site (Anova: p-value = 0.935, F-value = 0.205, Df = 4). While parasite richness was related to primate species (Anova: p-value = 0.014, F-value = 6.063, Df = 1), as well as to the study site (Anova: p-value:0.001, F-value = 4.506, Df = 4), (Tukey's test: S. cassiquiarensis vs. A. seniculus (0.014), Villavicencio vs. Cumaral (p-value:0.005)).

4. Discussion

Morphological analyses identified parasite taxa previously reported infecting NHPs in Latin America (Rondón et al., 2021; Solórzano-García and Pérez-Ponce de León, 2018), including the primate species here sampled, as well as new host records. For brown spider monkeys (A. hybridus), the protozoans Balantidium sp. and Entamoeba sp. have been previously reported (Solórzano-García and Pérez-Ponce de León, 2018), while Blastocystis sp., Giardia duodenalis, and Dientamoeba fragilis-like are new records. Similarly, for the varied white-fronted capuchin (C. versicolor) there are also new records: Blastocystis sp., Giardia sp., Eimeria sp., and Controrchis sp. For the gray-handed night monkey (A. griseimembra) and for the Humboldt's squirrel monkey (S. cassiquiarensis) all parasites here observed are new records. On the contrary, for the Colombian red howler monkey (A. seniculus) only Eimeria sp. and Dientamoeba fragilis-like are new records, and for the tufted capuchin (S. apella) the new records include Hymenolepis sp., Controrchis sp. and Ancylostomatidae.

Regarding the results obtained for one single primate species when sampled in different locations, S. cassiquiarensis was found infected by 14 parasite taxa. Interestingly, some parasites as Blastocystis sp. and Ascaris sp. were present only in Cumaral, the location where primates are in greater contact with humans and domestic animals, and are often fed by people. The effects of wildlife habitat loss and fragmentation due to human land usage (i.e. deforestation, crops, ecotourism) increase potential direct interactions between wildlife and humans as a consequence of an increased interface, and thus of an increased risk of transmission of zoonotic diseases from wildlife to humans, but also from humans to wildlife, as observed for the Ugandan mountain gorilla population and the prevalence of Giardia sp. infection which was considerably greater in the gorillas habituated to humans than the non-habituated gorillas (Patz et al., 2000).

In the case of A. seniculus, a higher number of parasite taxa was observed in a flooded forest (San Juan) rather than in terra firme forests (Maní and Yopal). For S. apella, the number of parasite taxa observed was equal in both sampling sites, Yopal surrounded by cattle ranching and Cabuyaro surrounded by palm oil plantations. However, Giardia sp. and Hymenolepis sp. were only present in samples from Yopal, while Ancylostomatidae and Controrchis sp. were present only in Cabuyaro. The presence of oil palm plantations is an ecological factor that might have an effect on parasite richness/prevalence by affecting the development of the eggs/larvae, as it is known that oil palm trees have different impacts, for instance in the local climate, tending to be hotter, drier and brighter than forests due to the less dense canopy (IUCN Oil Palm Task, 2018), thus negatively affecting the development of some parasite taxa. However, the weather conditions in terms of temperature and rainfall/humidity were still permissive for the development of parasites like trematodes and Ancylostomatidae, but it should be noted that the number of samples obtained in Cabuyaro was very low. For future studies it is advisable to increase the number of samples to better understand the parasite dynamics.

At San Juan, the study site with four sympatric primate species, differences in parasite taxa were observed according to NHP species. For instance, C. versicolor was infected with a higher number of parasite taxa, which could be explained by factors such as its omnivorous diet and the use of different forest strata, including the forest floor and understory, which could pose a risk of infection with soil-transmitted helminths, supported by the presence of Ancylostomatidae and strongyliform larvae.

In terms of molecular analyses, the identification of Giardia duodenalis provides new information from free-ranging NHPs. This parasite has previously been reported to infect free-ranging NHPs in the Americas: Alouatta pigra in Mexico (Bonilla-Moheno, 2002), Saimiri oerstedi and Cebus capucinus in Costa Rica (Appleton and Boinski, 1991; Parr et al., 2013), and Alouatta caraya in Argentina (Alegre et al., 2021; Kuthyar et al., 2021; Milozzi et al., 2012). In general, molecular studies on G. duodenalis infecting primates from Latin America are scarce, and only few of them had performed genotyping of the parasite, for example, in Brazil assemblage A was found in captive Alouatta clamitans (Volotão et al., 2008), and assemblages A and B in Ateles paniscus and Lagothrix cana, respectively, from the Wild Animal Rehabilitation Centre (Dos Reis et al., 2021), while in Argentina, assemblage B was found in A. caraya living in environments shared with humans, cattle, and dogs (Kuthyar et al., 2022). In Africa, Giardia has been reported infecting several species of NHPs and is considered to be a cause of morbidity, being humans and/or their livestock the likely source of infection in those NHPs according to molecular epidemiological investigations (Thompson, 2013). In Colombia, this is the first report of G. duodenalis assemblages A and B circulating in free-ranging Sapajus apella and Alouatta seniculus, respectively. Some of the samples here genotyped as assemblage B and one sample classified as assemblage A were collected in a forest fragment in Yopal, a city in the Casanare department. A previous study including human samples from Casanare reported assemblages A and B circulating in Pore city, and assemblage A circulating in Yopal city (Higuera et al., 2020).

In addition, records of Ascaris lumbricoides infecting NHPs in Colombia are of great interest because observations of this parasite infecting free-ranging primates are sporadic. It has been reported in free-ranging A. caraya and A. seniculus (Boero et al., 1968; Stuart et al., 1998), Alouatta palliata in Costa Rica (Stuart et al., 1990), and Ateles fusciceps in Panama (Stephens et al., 2017), as well as in captive Lagothrix lagothricha and A. seniculus in Peru and Colombia, respectively. Those previous records were based on morphology of the parasite, so are unable to distinguish between A. lumbricoides and Ascaris suum infection, as it is not possible to differentiate these species by their eggs (Leles et al., 2012). Thus, this is the first study using a molecular approach for identification of Ascaris at the species level from samples of American NHPs. In Colombia, the presence of A. lumbricoides circulating in pigs and human populations has been confirmed, as indicated by a recent study (Cavallero et al., 2021). In the present study, NHPs that were found infected with A. lumbricoides are in close contact with human populations, so this finding may suggest cross contamination between humans and NHPs.

For future research projects, several considerations could be recommended, such as the inclusion of environmental variables (e.g. temperature, humidity, forest cover, degree of forest fragmentation) and the performance of longitudinal sampling, which would allow more complete analyses in order to achieve a better understanding of the ecological dynamics of intestinal parasites infecting NHPs, as well as of the anthropic effects in fragmented forests.

5. Conclusions

The present study may help to develop a more accurate epidemiologic picture of intestinal parasites infecting free-ranging NHP species. Differences in parasite richness were found for some parasite taxa, primate species, and study sites, while parasite prevalence was not related to primate species or study sites. Factors such as diet, habits, environmental conditions, and interaction between humans and NHPs may have influenced the data obtained. For a better understanding of the molecular epidemiology of intestinal parasites, new surveys including humans and other NHP species, aiming to explore the distribution, genetic variation and host specificity are strongly recommended. In addition, educational activities with the exposed local communities should be promoted to increase the awareness of the potential risk of zoonotic transmission and the importance of avoiding food supply and physical contact with NHPs.

CRediT authorship contribution statement

Silvia Rondón: Writing – review & editing, Writing – original draft, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Serena Cavallero: Writing – review & editing, Writing – original draft, Supervision, Methodology, Conceptualization. Andrés Link: Writing – review & editing, Resources. Camila González: Writing – review & editing, Supervision. Stefano D'Amelio: Writing – review & editing, Supervision, Conceptualization.

Ethics statement

Ethical approval for the collection of faecal samples was obtained by Universidad de los Andes and the National Environmental Licensing Authority of Colombia (ANLA). Permits No: 2,020,006,799-1-000, 2021254742-1-000.

Funding

This research was supported by the Advances in Infectious Diseases, Microbiology, Legal Medicine and Public Health Sciences Doctorate of Sapienza University of Rome, and from Sapienza University of Rome trough the “Avvio alla Ricerca Tipo 2” (Protocol number: AR2221811FCCAA02) awarded to SR.

Declaration of competing interest

International Journal for Parasitology: Parasites & Wildlife.

With the present letter, we confirm no conflict of interest as authors of the original research article entitled “Intestinal parasites infecting free-ranging primates in Colombia: Morphological and molecular evidence” submitted to the journal. All authors have read and approved the final version of the manuscript.

Abbreviations

- IUCN

International Union for Conservation of Nature

- ML

Maximum Likelihood

- NHPs

Non-human primates

- TPI

Triosephosphate isomerase

Appendix A. Supplementary data

The following is/are the supplementary data to this article.