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.

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 ^4-7. 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.

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% 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).

Figure 1 Map of the study area for the collection of Didelphis marsupialis in Vereda El Alférez, Colombia. 

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).

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

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.

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. 

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 Leishmania⁶. 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 chagasi¹⁸ and T. cruzi¹⁹. 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. 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 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.