Molecular detection of the natural infection by trypanosomatid parasites in Didelphis marsupialis from a rural area in northern Colombia

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.


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 (1) .https://doi.org/10.17843/rpmesp.2023.401.11573Motivation 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.
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.
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.

KEY MESSAGES
In Colombia, more than 11 million people are estimated to be at risk of leishmaniasis, with 12,000 new cases reported annually since 2005 (2) .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) (3) .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 (4) .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 (4)(5)(6)(7) .
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 (4) .
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 (3,8) .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.

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% (9) .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) (9) .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. (8) .Each animal was implanted sub-dermally with a radio-frequency identification

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.

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.
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 (9) .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 (9,12,13) .These conditions add to the domestic animal husbandry, which would favor contact between hematophagous insects, potential reservoir mammals and parasites (13) .
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 (4,14,15) .In Latin America, this species has a high frequency of infection with leishmaniasis-causing agents (4) .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 (9) and also with Leishmania (6) .In some cases, as in Venezuela, D. marsupialis may have a Leishmania/T.cruzi coinfection (7,16) .
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 (15) .The weight of the specimens was similar to what was reported by Lozada et al. (17) .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) (16,17) .
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 (5,8) .
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 chagasi (18) and T. cruzi (19) .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 (18) .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 (9) .
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.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 (10) .
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 (15) , as well as its history as a reservoir in Colombia (6) and Venezuela (7,16) .
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.

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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 https://doi.org/10.17843/rpmesp.2023.401.11573

Figure 1 .
Figure 1.Map of the study area for the collection of Didelphis marsupialis in Vereda El Alférez, Colombia.Source: Marlon Mauricio Ardila.
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-ED-TA) buffer 5X, and visualized in Quantum-STA photo documentation system (Bio-Rad, California, USA®).
/doi.org/10.17843/rpmesp.2023.401.11573Positive 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.

Figure 2 .
Figure 2. Didelphis marsupialis specimen in a Tomahawk® trap collected in the village of El Alférez, Municipality of El Carmen de Bolívar, Colombia.

Figure 3 .
Figure 3. Agarose gel electrophoresis (1.5%-70 min/80 V) of 120 bp kinetoplast minicircle DNA (kDNA) reaction products from total blood of Didelphis marsupialis specimens, collected in rural area of the Municipality of El Carmen de Bolivar (Bolivar-Colombia).The 120 bp band corresponds to the amplified gene for the search of trypanosomatid parasites and the 289 bp band corresponds to the internal control of vertebrate DNA.