ABSTRACT

We studied the prevalence of infection by trypanosomatid parasites in Didelphis marsupialis and its relationship with morphological/age aspects in a rural area of El Carmen de Bolivar, Colombia. Five visits were made to the Vereda El Alférez; each of which lasted three consecutive nights. During these visits, Tomahawk® traps were installed in the peridomestic and wild ecotopes of the Vereda El Alférez. Body measurements, sex and age were determined from the collected animals. Blood was extracted by cardiopuncture, after sedation, in order to obtain total deoxyribonucleic acid (DNA) and amplify the conserved region of the kinetoplast minicircle DNA (kDNA) of parasitic trypanosomatids. The association between morphological parameters of didelphids and their frequency of infection by parasitic trypanosomatids was determined by binomial regression. Thirty D. marsupialis specimens (60.0% females and 40.0% males/66.7% adults and 33.3% juveniles) were collected. Molecular diagnosis revealed a frequency of trypanosomatid parasite infection of 46.7%. Stage (p=0.024) was a determinant for infection. We discuss the role of D. marsupialis as a potential reservoir of parasitic trypanosomatids in the Vereda El Alférez.

Keywords:
Leishmaniasis; Parasites; Didelphis; Colombia; DNA, Kinetoplast

KEY MESSAGES

1. Motivation for the study. A leishmaniasis outbreak in Vereda El Alférez, El Carmen de Bolívar, in the Colombian Caribbean, provided an opportunity to determine the presence of trypanosomatid parasites in cardiac blood of marsupials and the relationship of this infection with their morphological characteristics and age.
2. Main findings. The frequency of infection with trypanosomatid parasites was of 46.7% (14/30) among the collected marsupials. The developmental stage was the determining variable for infection.
3. Implications. The synanthropic habits of Didelphis marsupialis and the frequency of infection in cardiac blood by trypanosomatid parasites suggest it plays a role as a potential reservoir of parasites causing Chagas disease or leishmaniasis, which represents a risk for the emergence of new foci of zoonosis in the region.

Keywords:
Leishmaniasis; Parasites; Didelphis; Colombia; DNA, Kinetoplast

INTRODUCTION

Leishmaniasis is a neglected disease, and is very important for public health due to its high incidence and wide geographical distribution. According to the World Health Organization (WHO), leishmaniasis is found in 98 countries, with more than 12 million people infected with 20,000 to 30,000 deaths per year. More than 350 million people are estimated to be at risk of suffering from this type of zoonosis, with new cases ranging from 700,000 to one million, and mainly affecting populations living in areas with difficult access, lack of basic services, lack of education and low income ¹1. World Health Organization. Leishmaniasis [Internet]. Geneva: WHO; 2022 [citado el 10 de febrero de 2022]. Disponible en: https://www.who.int/news-room/fact-sheets/detail/leishmaniasis.
https://www.who.int/news-room/fact-sheet... .

In Colombia, more than 11 million people are estimated to be at risk of leishmaniasis, with 12,000 new cases reported annually since 2005 ²2. Instituto Nacional de Salud. Plan estratégico leishmaniasis 2018-2022 [Internet]. Bogotá: Ministerio de Salud, INS; 2022 [citado el 15 mayo de 2022]. Disponible en: https://www.minsalud.gov.co/sites/rid/Lists/BibliotecaDigital/RIDE/VS/PP/ET/plan-estrategico-leishmaniasis-2018-2022.pdf.
https://www.minsalud.gov.co/sites/rid/Li... . During 2019, 5082 cases were reported, of which 98.9% corresponded to cutaneous leishmaniasis (CL), 1.0% to mucocutaneous leishmaniasis (MCL) and 0.2% to visceral leishmaniasis (VL) ³3. Instituto Nacional de Salud. Informes de evento, período epidemiológico XIII. Colombia 2020 [Internet]. Bogotá: Ministerio de Salud, INS; 2022 [citado el 15 mayo de 2022]. Disponible en: https://www.ins.gov.co/Paginas/Inicio.aspx.
https://www.ins.gov.co/Paginas/Inicio.as... .

This zoonosis is caused by flagellates of the genus Leishmania (Kinetoplastea: Trypanosomatidae). The life cycle of these flagellates takes place in phlebotomine insects (Diptera: Psychodidae, Phlebotominae) that act as vectors; mammals of up to seven orders that act as reservoirs ⁴4. Roque AL, Jansen AM. Wild and synanthropic reservoirs of Leishmania species in the Americas. Int J Parasitol Parasites Wild. 2014;3(3):251-62. doi: 10.1016/j.ijppaw.2014.08.004.
https://doi.org/10.1016/j.ijppaw.2014.08... . Reports show that the frequency of Leishmania infection in rodents and marsupials is high, with Didelphis marsupialis (Didelphimorphia: Didelphidae) being the primary wild reservoir in several regions of Latin America, including Colombia ⁴4. Roque AL, Jansen AM. Wild and synanthropic reservoirs of Leishmania species in the Americas. Int J Parasitol Parasites Wild. 2014;3(3):251-62. doi: 10.1016/j.ijppaw.2014.08.004.
https://doi.org/10.1016/j.ijppaw.2014.08...

5. Corredor A, Gallego JF, Tesh RB, Morales A, Ferro C, Young DG, et al. Epidemiology of visceral leishmaniasis in Colombia. Am J Trop Med Hyg. 1989;40:480-86. doi: 10.4269/ajtmh.1989.40.480.
https://doi.org/10.4269/ajtmh.1989.40.48...

6. Travi BL, Jaramillo C, Montoya J, Segura I, Zea A, Goncalves A, et al. Didelphis marsupialis, an important reservoir of Trypanosoma (Schizotrypanum) cruzi and Leishmania (Leishmania) chagasi in Colombia. Am J Trop Med Hyg. 1994;50(5):557-65. doi: 10.4269/ajtmh.1994.50.557.
https://doi.org/10.4269/ajtmh.1994.50.55... ^-77. Viettri M, Herrera L, Aguilar CM, Morocoima A, Reyes J, Lares M, et al. Molecular diagnosis of Trypanosoma cruzi/Leishmania spp. coinfection in domestic, peridomestic and wild mammals of Venezuelan co-endemic areas. Vet Parasitol: Reg Stud Rep. 2018;14:123-30. doi: 10.1016/j.vprsr.2018.10.002.
https://doi.org/10.1016/j.vprsr.2018.10.... . The distribution of leishmaniasis in humans coincides with the distribution of vectors and potential reservoirs within the habitats they occupy, forming ecological systems that favor transmission cycles ⁴4. Roque AL, Jansen AM. Wild and synanthropic reservoirs of Leishmania species in the Americas. Int J Parasitol Parasites Wild. 2014;3(3):251-62. doi: 10.1016/j.ijppaw.2014.08.004.
https://doi.org/10.1016/j.ijppaw.2014.08... .

The municipality of El Carmen de Bolívar, located in Los Montes de María, northern Colombia, is one of the most important foci of VL in the country and has reported 325 human cases of leishmaniasis. Between 2008 and 2019, 75 cases of VL were reported in this region, which added to the presence of Lutzomyia evansi, one of the primary vectors ³3. Instituto Nacional de Salud. Informes de evento, período epidemiológico XIII. Colombia 2020 [Internet]. Bogotá: Ministerio de Salud, INS; 2022 [citado el 15 mayo de 2022]. Disponible en: https://www.ins.gov.co/Paginas/Inicio.aspx.
https://www.ins.gov.co/Paginas/Inicio.as... ^,88. Ardila MM, Carrillo-Bonilla L, Pabón A, Robledo SM. Surveillance of phlebotomine fauna and Didelphis marsupialis (Didelphimorphia: Didelphidae) infection in an area highly endemic for visceral leishmaniasis in Colombia. Biomédica. 2019;39(2):252-64. doi: 10.7705/biomedica.v39i2.3905.
https://doi.org/10.7705/biomedica.v39i2.... . Many cases of this zoonosis have been reported in Los Montes de María, however, there are few studies on wild reservoirs in this locality, mainly in the veredas, which are defined as rural nucleus close to an urban center with 50 to 1200 inhabitants. Therefore, the aim of this study was to evaluate the role of D. marsupialis as a potential reservoir of trypanosomatid parasites in this area of northern Colombia.

THE STUDY

Study area

The research was conducted in Vereda El Alférez, a rural area of the municipality of El Carmen de Bolívar (Department of Bolívar, 09º45’38.0” N - 075º10’19.1” W), located 200 meters above sea level (masl) with a temperature between 26-30 °C, bimodal rainfall and relative humidity between 75-85% ⁹9. Ardila MM, Herrera L, Zabala-Monterroza W, Bedoya-Polo A, Lozano-Arias D, García-Alzate R, et al. Molecular diagnosis of trypanosomatids in Didelphis marsupialis from Los Montes de María: a first report of Trypanosoma rangeli from Colombian Caribbean region. J Parasit Dis. 2021. doi: 10.1007/s12639-021-01459-x.
https://doi.org/10.1007/s12639-021-01459... . Human settlements in this area are located in a tropical dry forest, in which the inhabitants grow fruit trees, mainly cocoa, avocado and timber trees, both sources of economic activity in the region (Figure 1).

Collection and blood sampling of wild mammals

Five samplings were conducted from January 2018 to April 2019, using 21 Tomahawk® traps (66.04 cm × 22.86 cm × 22.86 cm) for three consecutive nights from 18:00 to 06:00 hours. Sampling was carried out in the peridomestic ecotope (PE), which corresponds to the surroundings of the dwelling (a diameter of up to 50 m), where domestic animals (poultry, pigs and cows) and synanthropic animals (rodents, marsupials and armadillos) can be found, as well as in the wild ecotope (S) (inside of the forest, 1 km from the PE) ⁹9. Ardila MM, Herrera L, Zabala-Monterroza W, Bedoya-Polo A, Lozano-Arias D, García-Alzate R, et al. Molecular diagnosis of trypanosomatids in Didelphis marsupialis from Los Montes de María: a first report of Trypanosoma rangeli from Colombian Caribbean region. J Parasit Dis. 2021. doi: 10.1007/s12639-021-01459-x.
https://doi.org/10.1007/s12639-021-01459... . Sampling both ecotopes required a total of 3024 hours.

The collected animals were taxonomically identified and then anesthetized for morphometric measurement, this included total length, tail length and leg length, as well as weight estimation; blood samples were also obtained following the procedure described by Ardila et al. ⁸8. Ardila MM, Carrillo-Bonilla L, Pabón A, Robledo SM. Surveillance of phlebotomine fauna and Didelphis marsupialis (Didelphimorphia: Didelphidae) infection in an area highly endemic for visceral leishmaniasis in Colombia. Biomédica. 2019;39(2):252-64. doi: 10.7705/biomedica.v39i2.3905.
https://doi.org/10.7705/biomedica.v39i2.... . Each animal was implanted sub-dermally with a radio-frequency identification (RFID) device for tracking and recording of their eventual recapture. The animals were released after recovering from anesthesia. Blood samples were labeled and refrigerated at -20°C for molecular analysis at the Biomedical Laboratory of the Biomedical Research Group of the University of Sucre. All specimens were legally collected in compliance with the “Permiso Marco de Recolectamiento de Especímenes de Especies Silvestres de la Diversidad Biológica con Fines de Investigación Científica No Comercial”, Resolution 0391 of April 11, 2016 issued by the Autoridad Nacional de Licencias Ambientales (ANLA), granted to the Universidad de Sucre.

Molecular detection of trypanosomatid parasites

The Wizard® Genomic DNA purification kit (Promega Corporation: Madison, Wisconsin, USA) was used for extracting total deoxyribonucleic acid (DNA) from blood samples, following the manufacturer’s instructions.

The NanoDrop 1000TM (Thermo Scientific, Massachusetts, USA) was used to quantify total DNA concentration. Molecular diagnostic was carried out by polymerase chain reaction (PCR) amplification of the highly repetitive sequence of kinetoplast minicircle DNA (kDNA) using primers 13A (5’-GGCCCACTATATTACACACCAACCAACCC-3’) and 13B (5’-GGGGGGTAGGGGGGCGTTCTCTGCGAA-3), which delimit a 120-bp fragment ¹⁰10. Rodgers MR, Popper SJ, Wirth DF. Amplification of Kinetoplast DNA as a Tool in the detection and diagnosis of Leishmania. Exp Parasitol. 1990;71:267-75. doi: 10.1016/0014-4894(90)90031-7.
https://doi.org/10.1016/0014-4894(90)900... . The primers XAHR 17 (5’-CGGAACCGCTCCTCATTGCC-3’) and XAHR 20 (5’-ACCCACACTGTGCCCATCTA-3’) were used for the detection of the vertebrate characteristic β-actin gene as internal control, which delimit a 289-bp fragment ¹¹11. Du Breuil RM, Patel JM, Mendelow BV. Quantitation of beta-actin-specific mRNA transcripts using xeno-competitive PCR. Genome Res. 1995;3(1):57-59. doi: 10.1101/gr.3.1.57.
https://doi.org/10.1101/gr.3.1.57... . DNA from Leishmania guyanensis was used as positive control and DNA from an infection-free opossum was used as negative control.

The PCR mix had a final volume of 23.0 µL of which 3.0 µL corresponded to blank DNA and the remaining 20 µL to the reaction mix, which contained 2.4 µL of PCR buffer (5X), 1.25 µL of Taq DNA polymerase (Thermo SCIENTIFIC), 0.18 µL of primers 13A and 13B, 0.12 µL of primers XAHR 17 and XAHR 20 and 5.16 µL of sterile high molecular grade milliQ water.

PCR was carried out on the Veriti 96 Well thermal cycler (Applied Biosystems, Carlsbad, USA®) under denaturing conditions at 95.0°C for 20 sec (40 cycles), alignment at 57, 6°C for 40 sec (40 cycles) and extension at 72°C for 5 min. The PCR products (7 µL) were subjected to horizontal electrophoresis (80 v for 70 min) in 1.5% agarose gel developed with ethidium bromide prepared in TBE (Tris-Borate-EDTA) buffer 5X, and visualized in Quantum-STA photo documentation system (Bio-Rad, California, USA®).

Positive samples revealed the presence of 120 bp bands by using the positive controls and a 1000 bp molecular size marker (Fermentas, Thermo Fisher Scientific®, Massachusetts, USA) as reference. Additionally, strains from species of the genus Trypanosoma: Trypanosoma cruzi and T. rangeli were assessed. The Primer-Blast tool (available online at the National Center for Biotechnology Information [NCBI]) was used to conduct a local alignment search to evaluate the specificity of the primers.

Data analysis

Descriptive statistics were used to analyze the abundance of mammals and the frequency of infection with trypanosomatid parasites. A binomial regression model (Statgraphics version 17) was used to assess the associations between the stage, sex and morphology of the collected mammals with the frequency of infection.

Ethical aspects

The study was approved by the Ethics Committee of the Universidad del Atlántico, Puerto Colombia, Colombia, with the code 21-10.

FINDINGS

Thirty specimens of D. marsupialis were collected (Figure 2), of which 66.7% (20/30) were adults and 33.3% (10/30) juveniles. Males accounted for 40.0% (12/30) and females for 60.0% (18/30), of which 33.3% (6/18) had pups in the marsupium at a rate of seven pups per female (Table 1).

On average, male specimens weighted more than females (1683g vs. 1138g), but females, on average, were longer than males (362mm vs. 244mm).

Molecular diagnosis of trypanosomatid parasites from the didelphids revealed an infection frequency of 46.7% (14/30), of which 57.0% (8/14) were adults and 43.0% (6/14) were juveniles (Figure 3). A 1:1 positivity ratio was found between males and females.

During the binomial regression analysis, we found that the only variable associated (p=0.024) with infection in individuals of D. marsupialis was the stage (8 adults and 6 juveniles), while the other variables such as sex (p=0.522), weight (p=0.131), total length (p=0.922), tail length (p=0.164) and leg length (p=0.074) showed values of p>0.05.

DISCUSSION

We searched for trypanosomatids in D. marsupialis, a synanthropic mammal, typical of biological corridors between wild and urban ecotypes, in the Vereda del Alférez (El Carmen de Bolívar, Northern Colombia). We collected and assessed 30 specimens, of which 46.7% (14/30) were infected by trypanosomatids. These findings strengthen the study of epidemiological scenarios in one of the most important leishmaniasis hotspots and a hypoendemic area for American trypanosomiasis ⁹9. Ardila MM, Herrera L, Zabala-Monterroza W, Bedoya-Polo A, Lozano-Arias D, García-Alzate R, et al. Molecular diagnosis of trypanosomatids in Didelphis marsupialis from Los Montes de María: a first report of Trypanosoma rangeli from Colombian Caribbean region. J Parasit Dis. 2021. doi: 10.1007/s12639-021-01459-x.
https://doi.org/10.1007/s12639-021-01459... .

Vereda de El Alférez is located in a wooded area, with small households and structural deficiencies, which facilitates the creation of a humid and warm environment that favors the entry of tropical disease vectors ⁹9. Ardila MM, Herrera L, Zabala-Monterroza W, Bedoya-Polo A, Lozano-Arias D, García-Alzate R, et al. Molecular diagnosis of trypanosomatids in Didelphis marsupialis from Los Montes de María: a first report of Trypanosoma rangeli from Colombian Caribbean region. J Parasit Dis. 2021. doi: 10.1007/s12639-021-01459-x.
https://doi.org/10.1007/s12639-021-01459... ^,1212. Linero JD, Cera-Vallejo Y, García-Alzate R, Herrera L, Ardila MM. Flebotominofauna y primer registro de Lutzomyia aclydifera (Diptera: Psychodidae) en el Caribe colombiano. Acta Biol Colomb. 2022;27(2):312-15. doi: 10.15446/abc.v27n2.91314.
https://doi.org/10.15446/abc.v27n2.91314... ^,1313. Posada-López L, Vélez-Mira A, Acosta LA, Cadena H, Agudelo D, Vélez ID. Descripción de un foco endémico de Leishmaniasis cutánea en Puerto Valdivia, Antioquia, Colombia. CES Salud Pública. 2014;5:3-10. doi: 10.21615/3029.
https://doi.org/10.21615/3029... . These conditions add to the domestic animal husbandry, which would favor contact between hematophagous insects, potential reservoir mammals and parasites ¹³13. Posada-López L, Vélez-Mira A, Acosta LA, Cadena H, Agudelo D, Vélez ID. Descripción de un foco endémico de Leishmaniasis cutánea en Puerto Valdivia, Antioquia, Colombia. CES Salud Pública. 2014;5:3-10. doi: 10.21615/3029.
https://doi.org/10.21615/3029... .

Didelphis marsupialis is a very eclectic animal in terms of habitats and is considered to be one of the synanthropic mastofauna indicative of human environmental impact ⁴4. Roque AL, Jansen AM. Wild and synanthropic reservoirs of Leishmania species in the Americas. Int J Parasitol Parasites Wild. 2014;3(3):251-62. doi: 10.1016/j.ijppaw.2014.08.004.
https://doi.org/10.1016/j.ijppaw.2014.08... ^,1414. Cabello DR. Reproduction of Didelphis marsupialis (Didelphimorphia: Didelphidae) in the Venezuelan Andes. Acta Theriol. 2006;51:427-33. doi: 10.1007/BF03195189.
https://doi.org/10.1007/BF03195189... ^,1515. Cruz-Salazar B, Ruiz-Montoya L, Navarrete-Gutiérrez D, Espinoza EE, Vázquez-Domínguez E, Vázquez L. Diversidad genética y abundancia relativa de Didelphis marsupialis y Didelphis virginiana en Chiapas, México. Rev Mex Biodivers. 2014;85:251-61. doi: 10.7550/rmb.36116.
https://doi.org/10.7550/rmb.36116... . In Latin America, this species has a high frequency of infection with leishmaniasis-causing agents ⁴4. Roque AL, Jansen AM. Wild and synanthropic reservoirs of Leishmania species in the Americas. Int J Parasitol Parasites Wild. 2014;3(3):251-62. doi: 10.1016/j.ijppaw.2014.08.004.
https://doi.org/10.1016/j.ijppaw.2014.08... . Particularly, in northern Colombia, these mammals have been found to be infected with T. cruzi, etiological agent of Chagas disease, with T. rangeli, which is infective for humans, but not pathogenic ⁹9. Ardila MM, Herrera L, Zabala-Monterroza W, Bedoya-Polo A, Lozano-Arias D, García-Alzate R, et al. Molecular diagnosis of trypanosomatids in Didelphis marsupialis from Los Montes de María: a first report of Trypanosoma rangeli from Colombian Caribbean region. J Parasit Dis. 2021. doi: 10.1007/s12639-021-01459-x.
https://doi.org/10.1007/s12639-021-01459... and also with Leishmania⁶6. Travi BL, Jaramillo C, Montoya J, Segura I, Zea A, Goncalves A, et al. Didelphis marsupialis, an important reservoir of Trypanosoma (Schizotrypanum) cruzi and Leishmania (Leishmania) chagasi in Colombia. Am J Trop Med Hyg. 1994;50(5):557-65. doi: 10.4269/ajtmh.1994.50.557.
https://doi.org/10.4269/ajtmh.1994.50.55... . In some cases, as in Venezuela, D. marsupialis may have a Leishmania/T. cruzi coinfection ⁷7. Viettri M, Herrera L, Aguilar CM, Morocoima A, Reyes J, Lares M, et al. Molecular diagnosis of Trypanosoma cruzi/Leishmania spp. coinfection in domestic, peridomestic and wild mammals of Venezuelan co-endemic areas. Vet Parasitol: Reg Stud Rep. 2018;14:123-30. doi: 10.1016/j.vprsr.2018.10.002.
https://doi.org/10.1016/j.vprsr.2018.10.... ^,1616. Herrera L, Morocoima A, Lozano-Arias D, García-Alzate R, Viettri M, Lares M, et al. Infections and coinfections by Trypanosomatid parasites in a rural community of Venezuela. Acta parasit. 2022. doi: 10.1007/s11686-021-00505-1.
https://doi.org/10.1007/s11686-021-00505... .

The ratio of three females for every two males of D. marsupialis found in the study area could be indicative of a low population size, given the possible existence of intra- and interspecific competition for resources and predation ¹⁵15. Cruz-Salazar B, Ruiz-Montoya L, Navarrete-Gutiérrez D, Espinoza EE, Vázquez-Domínguez E, Vázquez L. Diversidad genética y abundancia relativa de Didelphis marsupialis y Didelphis virginiana en Chiapas, México. Rev Mex Biodivers. 2014;85:251-61. doi: 10.7550/rmb.36116.
https://doi.org/10.7550/rmb.36116... .

The weight of the specimens was similar to what was reported by Lozada et al. ¹⁷17. Lozada S, Ramírez GF, Henry-Osorio JH. Características morfológicas de un grupo de zarigüeyas (Didelphis marsupialis) del suroccidente Colombiano. Rev Inv Vet Perú. 2015;26(2):200-05. doi: 10.15381/rivep.v26i2.11011.
https://doi.org/10.15381/rivep.v26i2.110... . The higher weight in males compared to females could be due to the reproductive period of females with higher energy expenditure, coinciding with lower water availability and higher solar irradiation during the sampling period (low precipitation) ¹⁶16. Herrera L, Morocoima A, Lozano-Arias D, García-Alzate R, Viettri M, Lares M, et al. Infections and coinfections by Trypanosomatid parasites in a rural community of Venezuela. Acta parasit. 2022. doi: 10.1007/s11686-021-00505-1.
https://doi.org/10.1007/s11686-021-00505... ^,1717. Lozada S, Ramírez GF, Henry-Osorio JH. Características morfológicas de un grupo de zarigüeyas (Didelphis marsupialis) del suroccidente Colombiano. Rev Inv Vet Perú. 2015;26(2):200-05. doi: 10.15381/rivep.v26i2.11011.
https://doi.org/10.15381/rivep.v26i2.110... .

The only variable associated with the frequency of trypanosomatid parasite infection in the population of D. marsupialis was the developmental stage (57.0% positive adults and 43.0% positive juveniles), showing that adults are more likely to be infected than juveniles, probably because adult males with nocturnal and nomadic habits are exposed to the vectors for much longer than juveniles, as are females with cubs that remain in burrows in sympatry with the hematophagous insects ⁵5. Corredor A, Gallego JF, Tesh RB, Morales A, Ferro C, Young DG, et al. Epidemiology of visceral leishmaniasis in Colombia. Am J Trop Med Hyg. 1989;40:480-86. doi: 10.4269/ajtmh.1989.40.480.
https://doi.org/10.4269/ajtmh.1989.40.48... ^,88. Ardila MM, Carrillo-Bonilla L, Pabón A, Robledo SM. Surveillance of phlebotomine fauna and Didelphis marsupialis (Didelphimorphia: Didelphidae) infection in an area highly endemic for visceral leishmaniasis in Colombia. Biomédica. 2019;39(2):252-64. doi: 10.7705/biomedica.v39i2.3905.
https://doi.org/10.7705/biomedica.v39i2.... .

In Colombia, studies related to the identification of trypanosomatid parasites in reservoirs mainly focus on domestic animals (mainly dogs) in constant direct interaction with humans and involved in the maintenance cycle of Leishmania chagasi¹⁸18. Paternina-Gómez M, Díaz Y, Paternina LE, Bejarano EE. Alta prevalencia de infección por Leishmania (Kinetoplastidae:Trypanosomatidae) en perros del norte de Colombia. Biomédica. 2013;33:375-82. doi: 10.7705/biomedica.v33i3.780.
https://doi.org/10.7705/biomedica.v33i3.... and T. cruzi¹⁹19. Cantillo-Barraza O, Garcés E, Gómez-Palacio A, Cortés LA, Pereira A, Marcet PL, et al. Ecoepidemiological study of an endemic Chagas disease región in northern Colombia reveals the importance of Triatoma maculata (Hemiptera: Reduviidae), dogs and Didelphis marsupialis in Trypanosoma cruzi maintenance. Parasites and Vectors. 2015;8:482. doi: 10.1186/s13071-015-1100-2.
https://doi.org/10.1186/s13071-015-1100-... . In Sincelejo, Sampués, and Oveja, municipalities in the Department of Sucre, northern Colombia, infection rates of 34.9%, 35.7%, and 11.1%, respectively, have been reported ¹⁸18. Paternina-Gómez M, Díaz Y, Paternina LE, Bejarano EE. Alta prevalencia de infección por Leishmania (Kinetoplastidae:Trypanosomatidae) en perros del norte de Colombia. Biomédica. 2013;33:375-82. doi: 10.7705/biomedica.v33i3.780.
https://doi.org/10.7705/biomedica.v33i3.... . No studies have been carried out in the study area searching for parasites of the genus Leishmania and Trypanosoma in canines.

Trypanosomatid parasites usually have macrophagotropism in “recruiter” tissues, therefore the fact that 46.7% of the specimens were positive for trypanosomatid parasites by cardiac blood PCR is unusual in leishmaniasis cases. This finding could be related to the presence of other trypanosomatid parasite species, such as T. cruzi and T. rangeli that have been reported in the area ⁹9. Ardila MM, Herrera L, Zabala-Monterroza W, Bedoya-Polo A, Lozano-Arias D, García-Alzate R, et al. Molecular diagnosis of trypanosomatids in Didelphis marsupialis from Los Montes de María: a first report of Trypanosoma rangeli from Colombian Caribbean region. J Parasit Dis. 2021. doi: 10.1007/s12639-021-01459-x.
https://doi.org/10.1007/s12639-021-01459... .

PCR results showed the amplification of the conserved region of the minicircle of the Leishmania species in the processed samples, however, a band of the same size also amplified in the strains of T. cruzi and T. rangeli evaluated later. Likewise, the results of the local alignment search carried out with Primer-Blast, showed that these primers also bind to such parasites, which contrasts with what is described in the literature for the specificity of this marker, thus broadening the pool of detectable organisms to other trypanosomatid parasites, but reducing the specificity of the test. The findings represent a risk factor for human populations since all these parasites are involved in the etiology of neglected tropical diseases transmitted by hematophagous insects, therefore, they should be taken into account for the design and evaluation of control strategies ¹⁰10. Rodgers MR, Popper SJ, Wirth DF. Amplification of Kinetoplast DNA as a Tool in the detection and diagnosis of Leishmania. Exp Parasitol. 1990;71:267-75. doi: 10.1016/0014-4894(90)90031-7.
https://doi.org/10.1016/0014-4894(90)900... .

The infection of D. marsupialis with trypanosomatids shows a dynamic equilibrium in the study area, which ensures the survival of the parasites, not only in time, but also regarding the geography, due to its high population growth rate, ecological eclecticism ¹⁵15. Cruz-Salazar B, Ruiz-Montoya L, Navarrete-Gutiérrez D, Espinoza EE, Vázquez-Domínguez E, Vázquez L. Diversidad genética y abundancia relativa de Didelphis marsupialis y Didelphis virginiana en Chiapas, México. Rev Mex Biodivers. 2014;85:251-61. doi: 10.7550/rmb.36116.
https://doi.org/10.7550/rmb.36116... , as well as its history as a reservoir in Colombia ⁶6. Travi BL, Jaramillo C, Montoya J, Segura I, Zea A, Goncalves A, et al. Didelphis marsupialis, an important reservoir of Trypanosoma (Schizotrypanum) cruzi and Leishmania (Leishmania) chagasi in Colombia. Am J Trop Med Hyg. 1994;50(5):557-65. doi: 10.4269/ajtmh.1994.50.557.
https://doi.org/10.4269/ajtmh.1994.50.55... and Venezuela ⁷7. Viettri M, Herrera L, Aguilar CM, Morocoima A, Reyes J, Lares M, et al. Molecular diagnosis of Trypanosoma cruzi/Leishmania spp. coinfection in domestic, peridomestic and wild mammals of Venezuelan co-endemic areas. Vet Parasitol: Reg Stud Rep. 2018;14:123-30. doi: 10.1016/j.vprsr.2018.10.002.
https://doi.org/10.1016/j.vprsr.2018.10.... ^,1616. Herrera L, Morocoima A, Lozano-Arias D, García-Alzate R, Viettri M, Lares M, et al. Infections and coinfections by Trypanosomatid parasites in a rural community of Venezuela. Acta parasit. 2022. doi: 10.1007/s11686-021-00505-1.
https://doi.org/10.1007/s11686-021-00505... .

A limitation of the study is that we did not characterize the species of circulating trypanosomatid parasites by using more specific genetic markers or sequencing. Limited funding contributed to this limitation.

In conclusion, the synanthropic behavior of these vertebrates, the frequency of infection with trypanosomatid parasites and the dynamic equilibrium between the parasite and the vertebrates imply the presence of rural cycles of zoonotic transmission that could eventually affect humans in Vereda El Alférez. Likewise, our results demonstrate the need for greater surveillance of vector-borne diseases, since these animals tend to be parasite amplifiers or transmission bridges between rural and urban cycles, representing an additional risk.

Acknowledgments.

The authors thank the community of El Alférez for their logistical support, as well as Alexander Bedoya-Polo for his support during molecular testing.

References

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