Trypanosoma spp. infection in urban and wild ecotopes of the caribbean region in Colombia

ABSTRACT We conducted a study to evaluate the frequency of infection by Trypanosoma spp. in bats captured in wild and urban ecotopes in the Department of Atlántico in the Caribbean region of Colombia from March 2021 to May 2022. Bats were taxonomically identified, and sex, relative age, and reproductive conditions were determined. A blood sample was used for parasitological analysis and DNA extraction to amplify a region of the 18S rRNA. 125 bats were collected, with the most abundant families being Molossidae (62/125; 49.6%) and Phyllostomidae (43/125; 34.4%). Molossus molossus collected in wild habitats showed an infection frequency of 8.1% (5/61) and 4.1% (3/61) through parasitological and molecular analysis, respectively. In comparison, Noctilio albiventris collected in urban habitats showed an infection frequency of 16.6% (2/12) for both analyses. These findings represent the first records of M. molossus harboring trypanosomes for the Department of Atlántico and of N. albiventris harboring trypanosomes in Colombia.


INTRODUCTION
Bats are hosts and reservoirs of several parasitic microorganisms (1) .Their ability to fly, longevity, mobility, and ecosystem services as seed dispersers, pollinators, and arthropod controllers place bats in the focus of eco-epidemiological surveillance for some zoonoses (2) .Some etiological agents of these zoonoses, such as trypanosomatids, including Trypanosoma and Leishmania (Euglenozoa: Kinetoplastea, Trypanosomatidae), are important in veterinary and human medicine (1,3) .
Trypanosoma cruzi, a parasite that causes Chagas disease (also known as American trypanosomiasis), affects people in Colombia.By 2023, there were 34 reported cases of acute Chagas disease (4)   .The way this disease spreads (transmission cycles) changes depending on the animals living in each area (ecotope).This shift is seen with T. cruzi and other similar parasites (trypanosomes).In some regions, the types of mammals that feed the disease-carrying insects (vectors) may influence the severity (virulence) and prevalence of the parasite (5) .https://www.doi.org/10.17843/rpmesp.2024.412.13598 Motivation for the study.The role of bats as hosts of Trypanosoma spp. in the Atlantic Department in Colombia, as well as its taxonomic diversity has been poorly studied.
Main findings.This is the first report of frequency of infection by Trypanosoma spp. in bats in the Atlántico Department in Colombia.
Implications.The great adaptive capacity of bats to different ecological niches and its role as hosts of Trypanosoma spp.for wild and urban ecotopes represents a risk factor in transmission cycles of epidemiological importance.

KEY MESSAGES
It has been investigated since 1982 that bats in Colombia may carry the Trypanosoma parasite.At that time, Marinkelle found T. cruzi in 233 bat blood samples, Trypanosoma cruzi marinkellei in 25 samples, and other Schizotrypanum species in 315 samples (6) .These infections were more common in central, eastern, and southern Colombia.Although bats from the Caribbean region were included in those studies, the frequency of Trypanosoma infection was not reported.That is why this study was designed to evaluate how frequently wild and urban bats in the Department of Atlántico carry Trypanosoma species and to understand their potential in the transmission of Chagas disease.

Capture, identification and marking of bats
We used two mist nets (12x2.5 meters, 3x3 cm mesh; BioWed®) to capture bats from March 2021 to May 2022.In both wild and urban areas, we conducted three sampling sessions over two consecutive nights (between 5:30 pm and 11:30 pm), totaling six sessions (12 nights) (8) .The total effort was 4,320 m 2 per hour, with an effort per night of 180 hours per network.
The taxonomic identification of the specimens was carried out with the keys of Díaz et al. (9) .Sex, age, and reproductive condition were determined following methods in Kunz et al. (10) .Microperforations were made in the left wing patagium to register recaptured.Another volume of blood was destined for parasitological analysis.The mor-phometric data for each animal were recorded on a self-prepared information sheet.

Parasitological and molecular diagnosis
Sterile blood (50 µL) was collected from the brachial vein.For subsequent molecular analysis, the blood was impregnated with Whatman filter paper and stored at 4°C.Each fresh blood sample was examined twice under an optical microscope (Leica Microsystems CMS GmbH, model DMi1, Morrisville, NC, USA) at 40X to search for haemoflagellates morphologically compatible with Trypanosoma and their possible quantification (11) .We used the Wizard® HMW purification kit (Promega) to extract DNA from blood samples soaked in filter paper, following the manufacturer's instructions.We then quantified the total DNA concentration using an EPOCH 2NS instrument (BioTeck Instruments).To detect Trypanosoma spp., we performed a nested PCR (Polymerase Chain Reaction) targeting a specific region of the 18S rRNA gene (positions 200-600).In the first PCR step, we used the primers TRY-927 forward and TRY-927 reverse.Positive samples generated a fragment of about 900 base pairs (bp) (12) .For the second PCR step, 5 µL of the first PCR product was used with the primers SSU-561 forward and SSU-561 reverse, amplifying a fragment between 320 and 560 bp (12) .To ensure the test's accuracy, we included a negative control (PCR mix without DNA) and a positive control (T.cruzi DNA, strain MDID/CO/2018/Dm006).
.The products obtained were evidenced by horizontal electrophoresis (100V/30 minutes) in 1.5% agarose gel, stained with Ethidium Bromide solution in TAE buffer, for 12 minutes, to be visualized and photo documented in the iBright™ imaging system FL1500 (Thermo Fisher Scientific Inc: MA, USA).
Samples were considered positive when a band between 320-560 bp was amplified (12) , using the 100-1,000 bp molecular size marker (MBiotech) as a reference.

Data analysis
The infection rate was estimated as a percentage, and the comparative analysis of the diversity of the number of bat species per location (q0/species richness; q1/typical species, and q2/ dominant species) was carried out based on abundance data according to Hill (13) .For this analysis, species rarefaction curves (Hill numbers) were performed using the "iNext" package in the R language (14) .A permutation test was performed in the PAST-4 program to look for significant differences between the diversity of order q0, q1, and q2 of bats captured in wild and urban locations.A Fisher's exact test was used to determine the association between the presence of Trypanosoma spp. in bats and ecotopes, and a chi-square test was used to determine the statistical differences between the infection rate in the collected bat species/ecotope.

Ethical aspects
The study was approved by the Ethics Committee of the Universidad del Atlántico, Puerto Colombia, Colombia, with the code 02-III-2021.

Richness, abundance and morphological characteristics of bats
A total of 125 bats were captured, belonging to four families and eight species, which were distributed in four trophic guilds (15) .The most abundant families were Molossidae (62/125, 49.6%) and Phyllostomidae (43/125, 34.4%), followed by Noctilionidae (18/125, 14.4%) and Vespertilionidae (2/125, 1.6%).Within the general external morphological characteristics of the bats studied, individuals of Molossus molossus (Figure 2A) exhibit a bicolor coat that is 4 mm in length, starting with a light base and transitioning to a reddish-brown.Additionally, the hair at the hip´s base measures 7.3 mm.These bats have an approximate weight of 18,8 g, an average forearm length of 40.1 mm, and a wingspan of 288.1 mm (15) .In addition, Noctilio albiventris individuals (Figure 2B) feature short, reddishyellow fur, weighs 26.7 g, and have a forearm length of 59,3 mm and a wingspan of 421.6 mm (15) .The species by area, the trophic guild, sex, relative age, and reproductive conditions to which they belong were complemented in Table 1.
The alpha diversity indices (Hill numbers) revealed a richness q0 for both areas, with the wild area presenting significantly higher richness than the urban area (p=0.010), even though the curve of rarefaction for the wild area is not stable.
The values of q1 for both zones revealed similar abundances but without significant differences (p=0.080), and both rarefaction curves tend to stabilize, a trend that is repeated for the values of q2, where it was observed a greater dominance in the assemblage of wild bats concerning the urban one, but without significant differences (p=0.080)(Figure 3).

Frequency of infection by Trypanosoma spp. in bats
Of the 125 captured bats, seven pregnant females were excluded (two from the wild ecotope and five from the urban ecotope), and 94.4% (118/125) of the bats were analyzed.Regarding the molecular diagnosis, it was obtained that 4.2% (5/118) of the samples amplified a band of 560 bp (Figure 4).In detail, M. molossus from the wild ecotope was found positive with 2.5% (3/118), while in the urban ecotope N. albiventris with 1.6% (2/118).
The frequency of infection by Trypanosoma spp.according to parasitological analyses for bats captured in wild ecotopes, it was 5.8% (5/86), and for urban ecotopes it was 6.2% (2/32).The bat species with the presence of sanguineous trypomastigotes were M. molossus (for wild ecotopes) and N. albiventris (for urban ecotopes).
No association was found between the presence of Trypanosoma spp. in bats and the ecotope (Fischer; p=1.000), as well as no significant differences between the frequency of infection by these parasites in bats from wild areas (2.5%) and those from urban areas (1.6%) (p=0.929).

DISCUSSION
Few studies have dealt with the biodiversity of the bat fauna of the Atlántico Department, and this is the first to contribute to updating the inventory of bats inhabiting assemblages of wild and urban ecotopes with tropical dry forest remnants, also including a first approximation of the presence of Trypanosoma spp. in these mammals.The present study covered 57.1% of the families reported in the Atlántico Department (16) and 44.4% of those recorded in the Neotropics (17).
When looking at the total number of bat species (q0), our data suggests a higher potential species richness in the wild ha-   fficient to capture a good representation of the most abundant bat species in each location.Interestingly, even though the urban area has fewer bat species overall, the distribution of those species appears to be more even (q2).However, this difference between wild and urban areas is not statistically significant (p=0.080).
The role of M. molossus as a trypanosomatids host has been reported in Venezuela by Añez et al. (11) , with evidence of congenital transmission of T. cruzi, and in Brazil by Oliveira da Silva (17) with 54.0% of infection frequency (7/13) for T.
cruzi and 69.0%(9/13) for Leishmania spp.For Colombia, the role of M. molossus as a host for trypanosomatids has been

Figure 1 .
Figure 1.Study area of the collection of bats in wild (red) and urban (blue) ecotopes of the Atlántico Department, Colombian Caribbean region.
bitat compared to the urban habitat.This indicates greater bat diversity in wild areas.The stable rarefaction curves for both wild and urban areas (q1) suggest our sampling effort was suhttps://www.doi.org/10.17843/rpmesp.2024.412.13598

Figure 3 .
Figure 3. Rarefaction curve for diversity of order q0 (species richness), q1 (typical species), and q2 (dominant species) of assemblages of bats collected in wild and urban ecotopes of the Atlántico Department, Colombian Caribbean region.

Table 1 .
Characteristics of bats captured in the Atlántico Department, Colombian Caribbean region.