Trypanosoma spp. and its association with mammals: a literature review and case studies for the Andes and Orinoquia regions of Colombia

M.J. Narváez-Moreno, L. Y. Mancilla-Agrono, E. T. Martínez-Sánchez, J. Alvarez-Londoño, P. A. Ossa-López, M. E. Álvarez-López, H. E. Ramírez-Chaves, F. A. Rivera-Páez*

María J. NARVÁEZ-MORENO, Biol, Programa de Biología, Facultad de Ciencias Exactas y Naturales, Universidad de Caldas, Manizales, Colombia; Lorys Y. MANCILLA-AGRONO, Biol, Estefani T. MARTÍNEZ-SÁNCHEZ, MSc, Doctorado en Ciencias – Biología, Facultad de Ciencias Exactas y Naturales, Universidad de Caldas, Manizales, Colombia; Johnathan ALVAREZ-LONDOÑO, MSc, Grupo de Investigación en Genética, Biodiversidad y Manejo de Ecosistemas (GEBIOME), Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad de Caldas, Manizales, Colombia; Paula A. OSSA-LÓPEZ, PhD, María E. ÁLVAREZ-LÓPEZ, MSc, Grupo de Investigación en Genética, Biodiversidad y Manejo de Ecosistemas (GEBIOME), Departamento de Ciencias Básicas de la Salud, Facultad de Ciencias para la Salud, Universidad de Caldas, Manizales, Caldas, Colombia; Héctor E. RAMÍREZ-CHAVES, PhD, Centro de Museos, Museo de Historia Natural, Universidad de Caldas, Manizales, Colombia; Fredy A. RIVERA-PÁEZ*, PhD (Corresponding author, e-mail: fredy.rivera@ucaldas.edu.co), Grupo de Investigación en Genética, Biodiversidad y Manejo de Ecosistemas (GEBIOME), Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad de Caldas, Manizales, Colombia

https://doi.org/10.46419/cvj.57.1.6

Abstract

Within Trypanosoma, some species are of public health concern, such as Trypanosoma cruzi which is the causative agent of Chagas disease, also known as American trypanosomiasis. This disease is endemic in 17 countries across the Americas and primarily affects mammals, including humans. In certain countries such as Colombia, there are still gaps in understanding the associations between Trypanosoma and mammals. In this context, this study aimed to consolidate the current state of knowledge regarding the association between Trypanosoma and mammals, while providing both morphological and molecular data on trypanosomes circulating in wild mammals in two departments of Colombia—Arauca and Caldas—located in the Orinoquia and Andes regions, respectively. To compile historical records of natural infections involving Trypanosoma species in wild and exotic mammals (introduced rats and mice) in Colombia, a literature review using the PRISMA method was conducted. To complement this, morphological and molecular detection and analysis were performed on samples from 109 wild mammals, including blood smears, organ impressions, and amplification of fragments of the mini-exon, Cytb, ITS, SSUrRNA, and gGAPDH genes. Additionally, a morphometric analysis of blood trypomastigotes was conducted. The review yielded 40 scientific articles that reported the presence of eight Trypanosoma species in wild and exotic mammals in Colombia, with the highest prevalence observed in bats and rodents. The analysis of samples from Arauca (n = 64) and Caldas (n = 45) revealed a Trypanosoma sp. prevalence of 37.6% (n = 41). These findings provide a comprehensive overview of the current knowledge on Trypanosoma and its association with mammals in Colombia. Furthermore, the results highlight the presence of Trypanosoma in different wild hosts in the Andes and Orinoquia regions, underscoring the need for enhanced research on wildlife within a One Health framework.

Key words: Public health; Systematic review; Trypanosomatida; Vector-borne diseases

Introduction

Approximately 60% of the emerging pathogens responsible for human illnesses are zoonotic, with wildlife serving as the primary reservoir for these pathogens (Brooks et al., 2014; Ramírez et al., 2014; Gonzalez-Astudillo et al., 2016; Ricardo-Caldera et al., 2024). The family Trypanosomatidae (Euglenozoa, Kinetoplastida) is represented by 19 genera, with Trypanosoma containing pathogenic species (Gruby, 1843; Kaufer et al., 2017). Trypanosoma sp. are responsible for parasitic diseases of medical and veterinary relevance (e.g., Trypanosoma brucei and Trypanosoma cruzi) (Rubio-Ortiz et al., 2020). Chagas disease or American trypanosomiasis is a zoonotic infection caused by T. cruzi, transmitted mainly by conenose bugs (Triatominae). This disease affects approximately six million people in the Americas and results in around 12,000 deaths per year (Bern and Montgomery, 2009; González-Rugeles, 2018; Pérez-Molina and Molina, 2018; World Health Organization, 2020; Ramos-Sesma et al., 2021). Trypanosoma cruzi circulates in different habitats, affecting both domestic and wild species (Jansen et al., 2020). In the wild cycle, triatomine species and wild mammal hosts are involved, while the domestic cycle includes triatomines, domestic and synanthropic mammals, and humans (Jansen et al., 2015; Rodríguez-Monguí et al., 2019).

The classification of Trypanosoma has been debated over the years. Hoare (1964) divided the genus into two sections: Stercoraria and Salivaria, differing in morphology, development, and vector transmission (Hoare, 1964; Simpson et al., 2006). Salivaria encompasses species whose vector development cycle ends in a salivary medium, resulting in continuous proliferation in the mammal host. This group contains the subgenus Trypanozoon, which includes the species that infects humans, Trypanosoma (Trypanozoon) brucei. Stercoraria includes species whose vector development cycle ends in faecal matter, leading to discontinuous proliferation in the mammal host. The Stercoraria group exhibits a wide range of species, forms, and life cycles, and includes the subgenus Schizotrypanum and contains one pathogen, Trypanosoma (Schizotrypanum) cruzi. (Chalmers, 1918; Hoare, 1964; Fraga et al., 2016; de Freitas et al., 2024; Kazim et al., 2024).

The life cycle of T. cruzi involves three distinct forms, morphologically and physiologically different in their mammal hosts and triatomines vectors: amastigotes, epimastigotes, and trypomastigotes (broad, slender or metacyclic) (Hoare, 1964; Tyler and Engman, 2001; De-Simone et al., 2022). The trypomastigotes found in mammalian blood are characterized by a “C” or “S” shape (length 16–25 μm), with a kinetoplast located at the posterior end, a free flagellum, and a waving membrane (De-Simone et al., 2022). Currently, T. cruzi is classified into seven discrete typification units (DTU): T. cruzi I (TcI), T. cruzi II (TcII), T. cruzi III (TcIII), T. cruzi IV (TcIV), T. cruzi V (TcV), T. cruzi VI (TcVI), and TcBat, which is specific to bats (Velásquez-Ortiz et al., 2022). These DTUs exhibit a histotropic clonal model in Chagas disease, where each DTU has a distinct tropism and is associated with different clinical manifestations of the disease (Macedo et al., 2002; Macedo and Segatto, 2010).

In Colombia, approximately one million people have been affected by Chagas disease, with an additional eight million at risk of contracting the infection (Oliveira et al., 2021; Gómez Ortega et al., 2022; Instituto Nacional de Salud, 2024). Trypanosoma cruzi infection has been detected in wildlife, domestic animals, and humans across the country (Oliveira et al., 2021; Gómez Ortega et al., 2022; Instituto Nacional de Salud, 2024). Several regions in Colombia are endemic for T. cruzi, including the Magdalena River valley, the Cundiboyacense high plateau, and the Catatumbo in the Andean region; the Sierra Nevada of Santa Marta in the Caribbean region, and the Llanos foothills and Serranía de la Macarena in the Orinoquia and Amazon regions. Among these, Orinoquia stands out as the endemic area with the highest mortality rate (Guhl, 2007; Oliveira et al., 2021; Gómez Ortega et al., 2022).

In Colombia, most studies on Trypanosoma have focused on humans and domestic mammals, with few examining wild mammals. Among the latter, the majority of the reports on T. cruzi infection have been associated with marsupials such as Didelphis marsupialis (Guhl and Ramírez, 2013; Reyes et al., 2017). Other mammalian orders identified as hosts include Cingulata (e.g., Dasypus novemcinctus), Rodentia (e.g., Akodon sp. and Dasyprocta spp.), Chiroptera (e.g., Carollia perspicillata and Artibeus obscurus), and Primates (e.g., Ateles spp. and Cebus spp.) (Jansen and Roque, 2010). Given that Colombia is recognised as one of the world’s biodiversity hotspots for mammal species, it is crucial to continue research efforts to enhance our understanding of the epidemiology scenario of T. cruzi across the different and diverse natural regions of the country. The objective of this study was to assess the current state of knowledge and provide new insights into the association between Trypanosoma and wild mammals in Colombia, with the aim of contributing to future control and prevention strategies within the framework of the One Health approach.

Figure 1. PRISMA diagram selection of articles for Colombia that summarises the sequence of selection of information for the case studies

Materials and Methods

Literature review of Trypanosoma associated with wild mammals in Colombia

To compile the available information on Trypanosoma in wild mammals and small, introduced, exotic commensal rodents (i.e., genera Mus and Rattus) in Colombia, a literature review was performed using the Scopus and Web of Science databases. The following key words combination were used in the search: “Trypanosoma AND mammals OR Tripanosomiasis AND mammals OR Chagas AND mammals”. The search was limited to scientific articles, with no restrictions on publication date or language.

The initial search yielded a total of 119 articles. After removing duplicates, 111 papers remained. The articles were reviewed to determine if they met the following inclusion criteria: 1) information on wild mammal species; 2) identification of the Trypanosoma species infecting the mammal host; 3) natural Trypanosoma infections, and 4) mention of the technique used to identify Trypanosoma (e.g., serological, molecular, or morphological). To minimise reviewer bias, the review was conducted by one person (MJNM) following the guidelines outlined in the PRISMA statement (Page et al., 2021). Ultimately, 40 articles met the inclusion criteria, of which 12 were obtained through free searches (Page et al., 2021) (Figure 1).

Table 1. Departments, municipalities and localities of wild mammals captured in the Andean and Orinoquia region of Colombian. Locality numbers are shown in Fig. 2

Detection and morphological, morphometric and molecular analysis of Trypanosoma associated with wild mammals in the Orinoquia and Andes regions of Colombia

To gather additional information on Trypanosoma and wild mammals in Colombia, field surveys were conducted in two departments (Figure 2) representing contrasting natural regions: Arauca (Orinoquia region) and Caldas (Andes region). In Arauca, samples were collected between October and November 2021, from different localities within the Arauca municipality (Table 1). This municipality has an agricultural, livestock, and oil exploitation-based economy (Rangel-Ch et al., 2017; Instituto de Hidrología Meteorología y Estudios Ambientales, 2019). In Caldas, samples were collected during March, September, and November 2022 (Figure 2; Table 1). The sampling localities in Caldas are located within the inter-Andean basin of the middle Magdalena and Cauca Rivers, at elevations ranging from 180 to 3903 m. This area supports coffee cultivation and livestock farming (Gobernación de Caldas, 2017; Cardona et al., 2018; Zapata-Torres, 2018), and is home to diverse ecosystems that provide a wide array of environmental services (Corporación Autónoma Regional de Caldas, 2020).capture bats (Chiroptera), four mist nets (12 x 2.5 m, mesh 15 mm) were set up. For terrestrial mammals, 60 Sherman and 10 Tomahawk traps were used, placed on the ground and above the vegetation at sampling locations. The traps were baited with sardines and a mixture of granola, banana, and vanilla essence (Voss and Emmons, 1996). Morphological measurements were taken to identify the captured mammals (e.g., tail length, ear, foot, and weight), and all animals were photographed. All animal handling procedures followed the guidelines of the Institutional Animal Care and Use Committee (Sikes et al., 2016; Institutional Animal Care and Use Committee Guidelines and Policies, 2018). Taxonomic identification was based on taxonomic keys proposed by Gardner (2008); Patton et al. (2015), and Díaz et al. (2021). Animal collection was conducted with a permit from the Autoridad Nacional de Licencias Ambientales (ANLA) granted to the Universidad de Caldas according to resolution N° 02497 of 31 December 2018, updated by resolution N° 000026 of 9 January 2024, and the Comité de Bioética de la Facultad de Ciencias Exactas y Naturales – Universidad de Caldas endorsement (2 June 2017 and 20 September 2019). No species registered in the wild threatened species of Colombian biological diversity, recorded in resolution N° 1912 of 2017, updated by resolution N° 0126 of 6 February 2024 were collected. All collected samples and specimens of interest were deposited in the Colección de Mamíferos, Museo de Historia Natural, Universidad de Caldas (MHN-UCa-M).

Figure 2. Study area a) South America; b) Colombia; c) Department of Caldas and d) Department of Arauca. The locality numbers are detailed in Table 1

Sample collection and morphological identification of Trypanosoma

For each captured mammal, a capillary or cardiac puncture blood sample was taken, with the skin sterilised beforehand using cotton soaked in 70% alcohol. A fraction of the blood was used to prepare blood smears (two slides per individual), while the remaining sample was stored in Eppendorf tubes with absolute ethanol for preservation and subsequent molecular analysis. In bats, capillary blood samples were obtained by puncturing the brachial vein, while for terrestrial mammals, such as marsupials and rodents, blood samples were obtained by cutting the posterior part of the tail (Cavazzana et al., 2010; Barbosa et al., 2017; Trujillo-Betancur, 2021). Cardiac puncture was performed immediately following euthanasia, following the Pan American Health Organization (2017) protocol, using a sterile 25G to 27G hypodermic needle, depending on animal size. To assess organ infection (i.e., spleen, brain, heart, liver, spinal cord, lungs, and kidney), organs of interest were extracted. Organ imprints were made on slides (Mancilla-Agrono et al., 2022). The organs were placed individually in sterile Petri dishes, washed with Phosphate Buffered Saline (PBS) (Farbehi et al., 2021), and then washed again with PBS. A longitudinal or transverse cut was made, excess blood was removed with sterile Wypall towels, and the organ was pressed several times onto the slide (multiple cuts were made when necessary). The organs were then stored individually stored in absolute ethanol at 4°C for subsequent molecular analysis. For paired organs, such as kidneys and lungs, a sample was taken from each organ.

Slides were fixed with absolute methanol for three minutes for blood smears and five minutes for organ imprints. They were then stained with 4% Giemsa solution for 40 minutes for blood smears and 45 minutes for organ imprints. Each slide was examined under a light microscope (Olympus BX43) at 100X magnification in bright field in the Molecular Biology Laboratory at the Universidad de Caldas.

Figure 3. Trypanosoma cruzi trypomastigote stage with the measurements used for classical morphometry

a) General structure of the trypanosomatids (kinetoplast, nucleus, undulating membrane and flagellum);
b) Measurements were taken for the distance between the following points: L = body length (with
flagellum); FF:L = flagellum free length; W = body width (at the middle of the core); BW = Maximum width
without membrane-bounded; MW = Maximum width membrane-bounded; CN = distance from the centre
of the kinetoplast to the centre of the nucleus; PK = distance from the posterior end of the body to the
centre of the kinetoplast, NK = distance from the centre of the kinetoplast to the front end; PN = distance
from the posterior end of the body to the centre of the nucleus; AN = distance from the forelimb of the
body to the centre of the nucleus; representative measures in bold (Modified from Hoare, 1972)

Morphological and morphometric analysis of Trypanosoma

Slide analyses were complemented by morphometric analysis of each Trypanosoma in the trypomastigote stage detected in blood smears and organ imprints. Measurements were taken following the guidelines of Dias and Freitas (1943), Hoare (1964 and 1972), and Abràmoff et al. (2004), using the ImageJ image analysis program (version 1.53t, 2022). A constant scale of 20 μm was set, and ten measurements were taken: total length (including the flagellum) (L), width over the nucleus (W), maximum width delimited by the membrane (MW), maximum width without delimitation by the membrane (BW), free flagellum length (FF), length of the kinetoplast with the anterior extremity (NK), distance from the kinetoplast to the posterior extremity (PK), distance to the nucleus [kinetoplast to nucleus] (CN), distance from the anterior extremity of the body to the centre of the nucleus (AN), and distance from the posterior extremity of the body to the centre of the nucleus (PN) (Figure 3).

The kinetoplast position index (KI) was calculated in relation to the nucleus and the posterior end of the body, the nucleus position index (NI) in relation to the posterior body end, and the flagellar index (FF:L), which defines the proportion of the free flagellum to body length (Pereira, 1965; Karbowiak and Wita, 2004; Barros et al., 2019). Principal Component Analysis (PCA) was performed using seven measurements (L, W, PK, NK, MW, BW, PN, and CN), before checking the normality for each of the variables using the Kolmogorov–Smirnov test (with Lilliefors correction), using the “lillie.test” v27.0 package to identify the variables that explained the greatest variability among the measurements and to visualise group formation in the morphometric space (Jolliffe, 2002; Balzarini et al., 2011). Analyses were conducted with the “cluster” v2.1.6, “NbClust” v3.0.1, and “vegan” v2.6-4 packages in RStudio 2023.03.1v (R Core Team, 2023).

Molecular Detection and Identification of Trypanosoma

DNA extraction from each tissue was performed using the Wizard® Genomic DNA Purification Kit (Promega Corporation) according to the manufacturer’s standard protocol. Pure water (negative control) and T. cruzi and Trypanosoma rangeli (positive controls, donated by the Laboratorio de Parasitología Tropical, Universidad del Tolima, Colombia) were used as controls for DNA extraction. The extracted DNA was subjected to PCR amplification of six fragments: 1) Kinetoplast DNA (kDNA); 2) intergenic region of the mini-exon gene; 3) Cytochrome b gene (Cyt b); 4) small subunit rRNA gene (SSUrRNA); 5) glyceraldehyde-3-phosphate dehydrogenase gene (gGAPDH); and 6) internal transcribed spacer (ITS) (Table S2). PCR reactions were performed in a final volume of 25 μL (6 μL 5X Buffer, 2.5 μL 10 mM dNTP mix, 0.5 μL each primer at 25 μM, 0.24 μL 5 U/μL Taq polymerase, and 3 μL DNA).

PCR products were visualised by horizontal electrophoresis in 1% agarose gels stained with ethidium bromide in a TBE 1X pH 8.0 running buffer at 100V/50mA, using a GelDoc-It®2310 Image photodocumenter (UVP), (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Some PCR products were purified using the Wizard® SV Gel and PCR Clean-Up Start-Up Kit (Promega Corporation). All amplification products, including purified ones, were sent for sequencing by Sanger’s method at Macrogen Inc. (South Korea). The obtained sequences were evaluated and edited using Genious Prime® v. 2022.2.2 software (Drummond et al., 2009), and species confirmation was performed using Nucleotide BLAST on the National Center for Biotechnology Information (NCBI).

Figure 4. Regions of Colombia in which studies on Trypanosoma have been conducted. The circles refer to the Trypanosoma species that have been reported in each region of Colombia, excluding the insular region where there are no records of Trypanosoma infection in wild mammals. The silhouettes of the animals represent the taxonomic groups of wild mammals in which infection by Trypanosoma spp. has been reported

Results

Literature review of Trypanosoma associated with wild mammals in Colombia

A total of 40 scientific articles met the inclusion criteria, encompassing 203 case studies involving 90 wild mammal species, grouped into 50 genera, 23 families, and eight orders across five regions of Colombia (Table 2, Figure 4). The literature review, covering studies up to August 2024, denotes the presence of eight Trypanosoma species in Colombia: T. cruzi, Trypanosoma dionisii, Trypanosoma evansi, Trypanosoma leeuwenhoeki, Trypanosoma minasense, T. rangeli, Trypanosoma theleiri, and Trypanosoma wauwau. In 46 of the case studies in six papers, the presence is reported of T. cruzi-bat, Trypanosoma sp.,

Table 2. Records of trypanosomes in Colombia from 1966 to 2024 with 203 case studies. For each department and species of Trypanosoma, the reference to the 40 articles is presented as a subscript. Authors of the articles indicate up to more than one species of Trypanosoma for a species.

Methods: a. Molecular techniques; b. Serological methods; c. Serodiagnosis; d. Morphological methods; e. Blood cultures; f. Morphometry; g. Histology

Marinkelle, 19661; Morales, et al., 19762; D’alessandro et al., 19843; Travi et al., 19894; Travi et al., 19945; Marquez et al., 19986; Falla et al., 20097; Rodríguez, et al., 20098; Cura et al., 20109; Farfán-García & Angulo-Silva, 201110; Mejía-Jaramillo et al., 201211; Gulh and Ramirez, 201312; Ramírez et al., 2013a13; Ramírez et al., 2013b14; Vásquez et al., 201315; Peña-García et al., 201416; Soto et al., 201417; Cantillo-Barraza et al., 201418; Mejía-Jaramillo et al., 201419; Ramírez et al., 201420; Cantillo-Barraza et al., 201521; León et al., 201522; Lima et al., 201523; Rendón et al., 201524; Gómez-Palacio et al., 201625; Messenger et al., 201626; Hernández et al., 201627; Reyes et al., 201728; Erazo et al., 201929; Gómez-Hernández et al., 201930; Wehrendt et al., 201931; Cantillo-Barraza et al., 202032; Erazo et al., 202033; Cantillo-Barraza et al., 202134; Patiño et al., 202135; Ardila et al., 202236; Cantillo-Barraza et al., 202237; Castillo-Castañeda et al., 202238; Ardila et al., 202339; Ricardo-Caldera et al., 202440

Figure 5. Regions of Colombia with reports of natural infection by Trypanosoma spp. for each mammal order. The inner circle indicates five of the six Colombian regions where cases of Trypanosoma studies in mammals have been reported; specifically, the outer circle shows the orders of wild and exotic mammals in which infections by some of the species have been reported of Trypanosoma

T.cruzi-like and T. rangeli-like species, as well as the Trypanosoma cruzi marinkellei (Table 2, Figure 4). The highest number of mammals infected by Trypanosoma species were from Chiroptera (28 species) and Rodentia (28 species), followed by Primates (14 species), Didelphimorphia (eight species), Cingulata (five species), Pilosa (four species), and Artiodactyla, Carnivora (two species) (Table 1). Chiroptera exhibited the greatest diversity of associated Trypanosoma species, including five species (T. cruzi, T. dionisii, T. evansi, T. rangeli, and T. theleiri), as well as T. c. marinkellei, T. cruzi-like, T. rangeli-like, and T. cruzi bat.

Seba’s short-tailed bat, Carollia perspicillata, showed the highest number of infections and Trypanosoma diversity, while the common opossum, Didelphis marsupialis, was reported in all articles as being infected by various Trypanosoma species. The common vampire bat, Desmodus rotundus, and the black rat, Rattus rattus, which were mentioned in 11 of the reviewed articles, with T. cruzi (Table 2) being the primary infection agent. Orders with fewer reports of T. cruzi or Trypanosoma sp. infection included Artiodactyla (1) and Cingulata (4) (Table 2).

The Amazon region presented exclusively T. cruzi infections, primarily in marsupials and xenarthrans such as Caluromys lanatus (Didelphimorphia), Dasypus novemcinctus (Cingulata), and Tamandua tetradactyla (Pilosa) (Figure 5, Table 2). In the Andes region, Chiroptera and Primates were the most affected orders, with higher infection rates in Phyllostomidae and Cebidae species (Figure 5, Table 2), caused by T. cruzi, T. cruzi bat, and T. rangeli. The Caribbean region showed a high prevalence of Trypanosoma infections in Rodentia families such as Cricetidae, Echimyidae, Heteromyidae, and Muridae (Figure 5, Table 2). In Didelphimorphia, infections by T. cruzi, T. rangeli, and T. wauwau were documented (Table 2). The Orinoquia region stood out for numerous reports of T. cruzi and T. cruzi-like lineage infections in wild mammals. The highest number of infections (Figure 5, Table 2) were recorded in Chiroptera (families Phyllostomidae and Vespertilionidae), followed by Rodentia (families Muridae and Cricetidae). In contrast, the Pacific region exhibited the highest incidence of infections in Rodentia and Didelphimorphia, primarily caused by T. cruzi, T. cruzi-like, T. leeuwenhoeki, and T. wauwau (Figure 5). PCR-based techniques were exclusively used in 60% (n = 24) papers for the detection of Trypanosoma spp., while 15% (n = 6) relied on serological methods. Additionally, 22.5% (n = 9) of the articles analysed used at least one of the following detection techniques: serodiagnosis, morphological methods, blood cultures, morphometry, or histology.

**Table 3. Mammals infected with Trypanosoma sp. in the departments of Arauca and Caldas, Colombia**

* New association between mammals and Trypanosoma sp. for Colombia a Organ infection location when there are more than two locations with infected individuals + Report of T. cruzi, strain Sylvio X10/1, TcI

Detection, morphological, morphometric and molecular analysis of Trypanosoma associated with wild mammals in two departments of Colombia

A total of 109 wild mammals were analysed including 45 from Caldas in the Andes region and 64 from Arauca in the Orinoquia region. The individuals studied belonged to four orders (Artiodactyla, Chiroptera, Didelphimorphia, and Rodentia), nine families (Caviidae, Cervidae, Cricetidae, Didelphidae, Emballonuridae, Noctilionidae, Molossidae, Phyllostomidae, and Vespertilionidae), and 46 species (Table S1). This study reports 20 new Trypanosoma associations for Colombia (Table 3), including the first report of T. c. marinkellei in the marsupial Metachirus myosuros, T. cruzi in the bat Orinoco serotine, Neoeptesicus orinocensis and Trypanosoma sp. in the common vampire-bat Desmodus rotundus.

The overall morphological or molecular prevalence of Trypanosoma was 37.6% (n = 41). Bat families with the highest prevalence of Trypanosoma were Phyllostomidae (52.4%, n = 22), Molossidae (33.3%, n = 10), and Vespertilionidae (16.7%, n = 3). In Arauca, a prevalence of 26.6% (n = 17) was recorded, with the following positive sample distribution: blood 53.3% (n = 16), liver and heart 13.3% (n = 4) each, and lung and kidney 10% (n = 3) each. In Caldas, the prevalence of Trypanosoma was 53.3% (n = 24), with the following positive sample distribution: blood 45.9% (n = 17), lung 16.2% (n = 6), liver and heart 13.5% (n=5) each, kidney 8.1% (n=3), and spleen 2.7% (n = 1) (Table 3). Trypomastigotes were identified morphologically in blood smears and impressions from liver, lung, and heart (Figures 6a – 6k). Coinfections with microfilariae were also observed in the blood of two bat species: Myotis handleyi and Phyllostomus discolor (Figures 6f and 6l).

The morphometric analysis showed variations in trypomastigote measurements to found on tissues and organs differents: Group 1 (G1) in blood (n = 4) and lung (n = 2) samples compared to Group 2 (G2) in lung (n = 3), heart (n = 1) and blood (n = 1) samples. These suggest variation both within and between groups based on classical morphometric parameters (Table S3). Forms of trypomastigotes observed included Slim “C” or “S” shaped forms (Figures S2a and S2b), as well as elongated intermediate and wide forms (Figures S2c, d, e). The position of the kinetoplast (Table S3, KI) varied; it was located at the posterior terminal end (KI < 2), near the nucleus (KI = 2), or in a subterminal position (KI > 2) (Figures S2e, f, and g). The nucleus was centrally located (NI = 1) (Figures S2a, b, e, f, h, and i), near the posterior end (NI < 1; Figure S2d), or near the anterior end (NI > 1; Figure S2g). A well-developed undulating membrane was observed in some trypomastigotes (Figures S2h, e, i), while others exhibited a flat membrane (Figures S2b and e). The length of the free flagellum (Table S3, FF:L) varied between long (Figures S2a, b, e, g, h, and i), short (Figures S2d and f), or absent (Figure S2c). The length of the free flagellum (Table S3, FF:L) varied between long (Figures S2a, b, e, g, h, and i), short (Figures S2d and f), or absent (Figure S2c). Eight of ten variables were selected using the Kolmogorov–Smirnov test (with Lilliefors correction) with normality significance (α= 0.05) (L, W, PK, NK, MW, BW, PN, and CN). The PCA indicated that the first two components explained 74.6% of the total variation, with PC1 accounting for 53.3% and PC2 contributing 21.3%. The analysis formed two groups (Figure S1), both dominated by bat species. In Group 1 (G1), trypomastigotes with the highest MW values were observed in the common vampire bat, D. rotundus (Table S3), while the rodent Microryzomys altissimus exhibited the lowest MW values (Table S3). In Group 2 (G2), the highest MW were found in N. orinocensis, while Artibeus lituratus exhibited the lowest values. Concerning the total length of Trypanosoma spp., morphotypes in G1 ranged between 9.3 μm and 16.04 μm, while total length in G2 ranged between 7.9 μm and 30.8 μm (Table S3). The kinetoplast position (PK) was smaller in G1 (0.7 μm and 3.8 μm) compared to G2 (1.6 μm and 7.5 μm). Additionally, the maximum width delimited by the membrane measurement (MW) was longer in G2 than in G1 (Table S3).

Two DNA sequences of Trypanosoma from N. orinocensis bats captured in the Orinoquia region were obtained (Table 3, Figure S3). The SSUrRNA gene sequences showed 100% identicality with T. cruzi, CL Brener strain, TcIIe [XM804040.1]. The sequence obtained for the gGAPDH gene showed 99.8% identicality with T. cruzi, matching the strain Sylvio X10/1, TcI [MG471429.1] (GenBank accession: PV147115).

Figure 6. Trypanosoma sp. in tissue samples from hosts from the departments of Arauca and Caldas.

Micrographs of trypomastigotes in mammal blood: a) Cynomops planirostris (Molossidae)-Arauca, b) Phyllostomus discolor (Phyllostomidae)-Caldas, c) Phyllostomus discolor (Phyllostomidae)-Arauca, d) Artibeus lituratus (Phyllostomidae)-Caldas, e) Odocoileus v. cariacou (Cervidae)-Caldas, f) Myotis handleyi (Vespertilionidae), trypomastigote (arrow) and coinfection with microfilaria (round arrow)-Arauca. Micrographs of trypomastigotes in organs: g) Neoeptesicus orinocensis (Vespertilionidae) in liver-Arauca, h) Dermanura phaeotis (Phyllostomidae) in lung-Caldas, i) lung of Artibeus lituratus (Phyllostomidae)-Caldas, j) Microryzomys altissimus (Cricetidae) in liver-Caldas, k) Platyrrhinus helleri (Phyllostomidae) in heart-Caldas, l) Phyllostomus discolor (Phyllostomidae), trypomastigote (arrow) with coinfection with microfilaria (round arrow)-Arauca. Bar = 20 μm

Table S1. Primers and amplification conditions for each of the genes used for the detection and identification of Trypanosoma spp. in two departments of Colombia

Table S2. Wild mammals sampled in the Departments of Arauca and Caldas, Colombia

Figure S2. Micrographs of the trypomastigote stage of Trypanosoma spp. in wild mammals from Colombia.

Table S3. Measurements of Trypanosoma sp. trypomastigote stage for classical morphometry. Measurements of group G1 are shown in blue and the measurements of group G2 obtained from principal component analysis (PCA) are shown in orange

Total length (L), width over the nucleus (W), maximum width delimited by the membrane (MW), maximum width without delimitation by the membrane (BW), length of the free flagellum (FF), length of the kinetoplast with anterior end (NK), distance of the kinetoplast with posterior end (PK), distance kinetoplast to nucleus (CN), distance anterior end of the body to the center of the nucleus (AN), distance posterior end of the body to the center of the nucleus (PN), ratio of the position of the kinetoplast (KI) relative to the nucleus and to the posterior end of the body; position of the nucleus (NI) relative to the posterior end of the body, and the flagellar index (FF:L) which defines the proportion of the free flagellum to the body length.

Figure S1. Principal Component Analysis (PCA).

Sample Trypanosoma cruzi trypomastigotes isolated from wild mammals. Group 1 (G1) species: Neoeptesicus orinocensis (blood), Microryzomys altissimus (lung), Molossus pretiosus (blood), Desmodus rotundus (blood) and Phyllostomus discolor (1-blood and 2-lung). Group 2 (G2) species: Artibeus lituratus (heart), Dermanura phaeotis (1-lung and 2-lung), Neoeptesicus orinocensis (2-lung) and Odocoileus virginianus cariacou (blood)

Discussion

The results reveal that in Colombia, wild mammals and commensal rodents are widely infected with Trypanosoma species known for their zoonotic relevance (Coura and Dias, 2009; World Organization for Animal Health, 2021). However, the presence of Trypanosoma spp. has primarily been documented in human infections, with T. cruzi showing the highest number of records (Rodríguez- Monguí et al., 2019). As a result, research on Trypanosoma in wild mammals remains limited, and the epidemiological status of trypanosomes infecting mammals endemically is poorly understood (Vásquez et al., 2013).

Records of Trypanosoma spp. infections in Colombia are scarce, especially when compared to studies from six biomes of Brazil (Jansen et al., 2018, 2020) and other countries (Fetene et al., 2021), where bats are considered amplifying hosts for various Trypanosoma species (da Silva et al., 2009; Matiz-González et al., 2025). The Trypanosoma species documented in Colombia include T. cruzi, T. c. marinkelei, T. dionisii, and T. rangeli, which are also documented in other countries of the Americas (Cavazzana, et al., 2010; Hamilton et al., 2012; Lima et al., 2015; Pinto et al., 2015; Bento et al., 2018; Barros et al., 2019). In particular, the bat family Phyllostomidae, the most diverse bat family in the Neotropical region (Ramírez-Chaves et al., 2024), plays a crucial role in the zoonotic cycles of these Trypanosoma species, with prior reports from Panama, Colombia, Ecuador, and Brazil (Hoare, 1972; Cottontail et al., 2014; Ramírez et al., 2014; Pinto et al., 2015), Similarly, associations between Didelphidae and Trypanosoma have been more frequently documented in Brazil, the United States, and Mexico (Sánchez-Cordero et al., 2024). Species such as D. marsupialis (Didelphimorpha) stand out as the key reservoir of T. cruzi, with circulating forms of the parasite in peripheral blood and infective forms in its anal glands (Tineo-González et al., 2023). These marsupials play an active role in the zoonotic transmission cycles of Trypanosoma and are considered an excellent reservoir due to high rates of persistent infections (Noireau et al., 2009; Herrera, 2010; Rodríguez-Monguí et al., 2019; Ardila et al., 2023). It is emphasised that for the Orinoquia region, the species Myotis brandtii Eversmann, 1845 (Chiroptera, Vespertilionidae) is not present in Colombia, although it was referenced in an earlier study (Castillo-Castañeda et al., 2022). This species is mainly documented in Europe (Taake, 1984; Piksa et al., 2022). Another taxon reported for the Orinoquia as Rattus spinosus (Rodentia) (Gulh and Ramirez, 2013) does not exist and we included as Rodentia.

In Colombia, most studies have concentrated on endemic regions of Chagas disease, such as the Orinoquia region (departments of Casanare and Meta) and the Caribbean region (departments of Bolívar and Magdalena) (Oliveira et al., 2021; Gómez Ortega et al., 2022). While these areas have been the main focus of research due to their high prevalence, there remains a significant gap in studies from other regions, such as the Insular, Amazon and Pacific regions (Figure 4, Table 2). Expanding research encompasses these regions to better understand the circulation of Trypanosoma spp. within different ecosystems (Moncayo, 2003), and investigate whether natural reservoirs and triatomine vectors (e.g., Triatoma dimidiata, Rhodnius prolixus and Panstrongylus geniculatus) are present (Méndez-Cardona et al., 2022), providing valuable insights for generating a more comprehensive epidemiological outlook.

The analyses conducted on the presence of Trypanosoma in wild mammals from the Andes and Orinoquia regions of Colombia, based on field data, provided morphological and molecular evidence of the presence of T. cruzi. Additionally, T. c. marinkellei infection is reported for the first time in the marsupial Metachirus myosuros, and T. cruzi in the bat N. orinocensis. These findings underscore the significant role of marsupials and bats in the circulation of Trypanosoma spp., as previously suggested (Travi et al., 1994; Castillo-Castañeda et al., 2022; Ricardo-Caldera et al., 2024). Furthermore, bats are known as potential reservoirs of Chagas disease in Colombia, with an infection rate higher than those observed in other mammals (Jansen et al., 2018).

According to our morphological analyses, T. cruzi shows pleomorphisms (Brener and Chiari, 1963; Schottelius and Uhlenbruck, 1983). This is evident in metacyclic trypomastigotes, whose length can range from 12.03 ± 3.03 μm as the smallest size, reaching between 26–34 μm when including the free flagellum. These trypomastigotes show notable differences in shape, size, and position (Navarro et al., 2003; Trujillo-Betancur, 2021) (Figure S2, Table S3). In natural infections, the manifestation of T. cruzi can vary, with more slender forms being predominant, and having greater tissue penetration capacity (Schottelius and Uhlenbruck, 1983; Beatty et al., 2024). However, this morphological variability complicates the establishment of a standardised measurement of trypomastigotes, as T. cruzi often disperses multifocally in tissues, resulting in significant polymorphism in the peripheral blood of the mammalian host (Rimoldi et al., 2012; Kunii et al., 2022). Since these parasites undergo a transformation process from epimastigotes, intracellular parasites do not differentiate synchronously, allowing for the observation of all transition stages in a single cell (Souza, 2002). Therefore, in diagnostics, it is crucial to consider factors like the infection stage, site, development stages of the parasite, host variability, and immune responses (Hoare, 1972).

This study represents the first morphological and molecular report confirming the presence of T. cruzi strains CL Brener, TcIIe, and Sylvio X10/1, TcI in two individuals of the bat N. orinocensis. This aligns with reports in other bat species, highlighting their ability to harbour T. cruzi DTUs, especially TcI and TcII, as well as representatives of the subgenus Schizotrypanum, such as T. c. marinkellei, which are exclusive to bats in the Americas (Amórtegui et al., 2022). Furthermore, the association of Trypanosoma sp. in the common vampire bat D. rotundus aligns with previous studies (Villena et al., 2018; Bergner et al., 2021), which detected T. cruzi in the salivary glands and identified the TcI lineage of T. cruzi in other vampire bat species like Diphylla ecaudata, and Diaemus youngii. The findings underscore the important role of bats in the transmission of T. cruzi infection to humans (Lazo et al., 2019; Bergner et al., 2020; Acosta-Jamett and Chaves, 2024). It is known that TcI primarily affects humans and can lead to Chagas cardiomyopathy, meningoencephalitis in immunocompromised patients, and affect the central nervous system (Miles et al., 2009; Shelton and Gonzáles, 2024). In this context, bats could serve as both reservoirs and transmitters of the parasite to other mammals, including humans, even in absence of triatomine vectors (Bustamante et al., 2014; Jansen et al., 2015, 2020; Villena et al., 2018). Therefore, it is essential to enhance studies involving wildlife under the One Health framework to better understand the epidemiology and transmission dynamics of T. cruzi.

Acknowledgments

We thank all members of the GEBIOME research group, the Molecular Biology Laboratory and members of the Natural History Laboratory, Natural History Museum, Universidad de Caldas, Manizales, Colombia, particularly Dr. Erika M. Ospina-Pérez, for their valuable collaboration and support during the development of this research. We also extend our gratitude to the Tropical Parasitology Research Laboratory, Universidad del Tolima, for donating the positive controls of Trypanosoma spp.

Funding

This project was funded by the program “Relación, distribución, taxonomía de especies de garrapatas asociadas a mamíferos silvestres en zonas endémicas de rickettsiosis en Colombia. Un ac ercamiento a la comprensión de la relación vectores patógenos-reservorio” [Code 120385270267]. Project: Garrapatas asociadas a mamíferos silvestres en el departamento de Caldas: Diversidad, detección de patógenos y distribución. Funded by: Minciencias; [Code: 71717]. Project: Convocatoria de Apoyo con Recursos Económicos a Grupos De Investigación De La Universidad de Caldas Año 2024. Funded by: Vicerrectoría de investigaciones y Posgrados – Universidad de Caldas.

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Trypanosoma spp. i njihova povezanost sa sisavcima: pregled literatura i slučajeva za regije Ande i Orinoquía u Kolumbiji

María J. NARVÁEZ-MORENO, Biol, Programa de Biología, Facultad de Ciencias Exactas y Naturales, Universidad de Caldas, Manizales, Colombia; Lorys Y. MANCILLA-AGRONO, Biol, Estefani T. MARTÍNEZ-SÁNCHEZ, MSc, Doctorado en Ciencias – Biología, Facultad de Ciencias Exactas y Naturales, Universidad de Caldas, Manizales, Colombia; Johnathan ALVAREZ-LONDOÑO, MSc, Grupo de Investigación en Genética, Biodiversidad y Manejo de Ecosistemas (GEBIOME), Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad de Caldas, Manizales, Colombia; Paula A. OSSA-LÓPEZ, PhD, María E. ÁLVAREZ-LÓPEZ, MSc, Grupo de Investigación en Genética, Biodiversidad y Manejo de Ecosistemas (GEBIOME), Departamento de Ciencias Básicas, Facultad de Ciencias para la Salud, Universidad de Caldas, Manizales, Caldas, Colombia; Héctor E. RAMÍREZ-CHAVES, PhD, Centro de Museos, Museo de Historia Natural, Universidad de Caldas, Manizales, Colombia; Fredy A. RIVERA-PÁEZ, PhD, Grupo de Investigación en Genética, Biodiversidad y Manejo de Ecosistemas (GEBIOME), Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad de Caldas, Manizales, Colombia.

Unutar roda Trypanosoma, vrste kao Trypanosoma cruzi predstavljaju javnozdravstveni problem. T. cruzi je uzročnik Chagasove bolesti, poznate i kao američka tripanosomijaza. Navedena je bolest endemična u 17 zemalja diljem Amerika i prije svega pogađa sisavce, uključujući ljude. U nekim zemljama kao što je Kolumbija povezanosti Trypanosoma i sisavaca još je nedovoljno poznato. U tome kontekstu, cilj je ovoga istraživanje sakupljati sadašnja znanja o povezanosti Trypanosoma i sisavaca, navodeći i morfološke i molekularne podatke o tripanosomima u divljim sisavcima u dvije Kolumbijske regije – Arauca i Caldas – koje se nalaze u Orinoquiji, odnosno u Andskoj regiji. Kako bi se prikupilo povijesne evidencije prirodnih zaraza vrstama Trypanosoma u divljih i egzotičnih sisavaca (uneseni štakori i miševi) u Kolumbiji, proveden je literaturni pregled korištenjem metode PRISMA. Komplementarno navedenome, izvelo se morfološko i molekularno analiza uzoraka od 109 divljih sisavaca, uključujući krvne briseve, otiske organa i pojačavanje fragmenata mini-eksona, Cytb, ITS, SSUrRNA i Ggapdh gena. Dodatno se provela morfometrijska analiza krvnih tripomastigota. Pregled literature pronašao je 40 znanstvenih članaka koji su navodili prisutnost osam vrsta Trypanosoma u divljih i egzotičnih sisavaca u Kolumbiji, s najvišom prevalencijom u šišmiša i glodavaca. Analiza uzoraka iz Arauce (n=64) i Caldasa (n=45) otkrila je prevalenciju vrste Trypanosoma od 37,6 % (n=41). Navedeni rezultati čine sveobuhvatni pregled sadašnjeg znanja o vrstama Trypanosoma i njihovim povezanostima sa sisavcima u Kolumbiji. Nadalje, rezultati ističu prisutnost Trypanosoma u različitim divljim domaćinima u regijama Anda i Orinoquije, naglašavajući potrebu za pojačanim istraživanjem divljih životinja u okviru programa One Health.

Ključne riječi: javno zdravstvo, literaturni pregled, Tripanosomatide, vektorski prenosive bolesti

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Trypanosoma spp. and its association with mammals: a literature review and case studies for the Andes and Orinoquia regions of Colombia

Abstract

Introduction

Figure 1. PRISMA diagram selection of articles for Colombia that summarises the sequence of selection of information for the case studies

Materials and Methods

Table 1. Departments, municipalities and localities of wild mammals captured in the Andean and Orinoquia region of Colombian. Locality numbers are shown in Fig. 2

Detection and morphological, morphometric and molecular analysis of Trypanosoma associated with wild mammals in the Orinoquia and Andes regions of Colombia

Figure 2. Study area a) South America; b) Colombia; c) Department of Caldas and d) Department of Arauca. The locality numbers are detailed in Table 1

Sample collection and morphological identification of Trypanosoma

Figure 3. Trypanosoma cruzi trypomastigote stage with the measurements used for classical morphometry

Morphological and morphometric analysis of Trypanosoma

Molecular Detection and Identification of Trypanosoma

Results

Table 2. Records of trypanosomes in Colombia from 1966 to 2024 with 203 case studies. For each department and species of Trypanosoma, the reference to the 40 articles is presented as a subscript. Authors of the articles indicate up to more than one species of Trypanosoma for a species.

**Table 3. Mammals infected with Trypanosoma sp. in the departments of Arauca and Caldas, Colombia**

Detection, morphological, morphometric and molecular analysis of Trypanosoma associated with wild mammals in two departments of Colombia

Figure 6. Trypanosoma sp. in tissue samples from hosts from the departments of Arauca and Caldas.

Table S1. Primers and amplification conditions for each of the genes used for the detection and identification of Trypanosoma spp. in two departments of Colombia

Table S2. Wild mammals sampled in the Departments of Arauca and Caldas, Colombia

Figure S2. Micrographs of the trypomastigote stage of Trypanosoma spp. in wild mammals from Colombia.

Table S3. Measurements of Trypanosoma sp. trypomastigote stage for classical morphometry. Measurements of group G1 are shown in blue and the measurements of group G2 obtained from principal component analysis (PCA) are shown in orange

Figure S1. Principal Component Analysis (PCA).

Discussion

Acknowledgments

Funding

Trypanosoma spp. i njihova povezanost sa sisavcima: pregled literatura i slučajeva za regije Ande i Orinoquía u Kolumbiji

Catarina JOTA BAPTISTA

Abstract

Introduction

Figure 1. PRISMA diagram selection of articles for Colombia that summarises the sequence of selection of information for the case studies

Materials and Methods

Table 1. Departments, municipalities and localities of wild mammals captured in the Andean and Orinoquia region of Colombian. Locality numbers are shown in Fig. 2

Detection and morphological, morphometric and molecular analysis of Trypanosoma associated with wild mammals in the Orinoquia and Andes regions of Colombia

Figure 2. Study area a) South America; b) Colombia; c) Department of Caldas and d) Department of Arauca. The locality numbers are detailed in Table 1

Sample collection and morphological identification of Trypanosoma

Figure 3. Trypanosoma cruzi trypomastigote stage with the measurements used for classical morphometry

Morphological and morphometric analysis of Trypanosoma

Molecular Detection and Identification of Trypanosoma

Results

Table 2. Records of trypanosomes in Colombia from 1966 to 2024 with 203 case studies. For each department and species of Trypanosoma, the reference to the 40 articles is presented as a subscript. Authors of the articles indicate up to more than one species of Trypanosoma for a species.

Table 3. Mammals infected with Trypanosoma sp. in the departments of Arauca and Caldas, Colombia

Detection, morphological, morphometric and molecular analysis of Trypanosoma associated with wild mammals in two departments of Colombia

Figure 6. Trypanosoma sp. in tissue samples from hosts from the departments of Arauca and Caldas.

Table S1. Primers and amplification conditions for each of the genes used for the detection and identification of Trypanosoma spp. in two departments of Colombia

Table S2. Wild mammals sampled in the Departments of Arauca and Caldas, Colombia

Figure S2. Micrographs of the trypomastigote stage of Trypanosoma spp. in wild mammals from Colombia.

Table S3. Measurements of Trypanosoma sp. trypomastigote stage for classical morphometry. Measurements of group G1 are shown in blue and the measurements of group G2 obtained from principal component analysis (PCA) are shown in orange

Figure S1. Principal Component Analysis (PCA).

Discussion

Acknowledgments

Funding

Trypanosoma spp. i njihova povezanost sa sisavcima: pregled literatura i slučajeva za regije Ande i Orinoquía u Kolumbiji

Catarina JOTA BAPTISTA

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**Table 3. Mammals infected with Trypanosoma sp. in the departments of Arauca and Caldas, Colombia**