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.

Keywords:
Colombia; bats; Trypanosoma; Zoonoses

KEY MESSAGES

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.

Keywords:
Colombia; bats; Trypanosoma; Zoonoses

INTRODUCTION

Bats are hosts and reservoirs of several parasitic microorganisms ¹1. Jaimes-Dueñez J, Cantillo-Barraza O, Triana-Chavez O, Mejia-Jaramillo AM. Molecular surveillance reveals bats from eastern Colombia infected with Trypanosoma theileri and Trypanosoma wauwau-like parasites. Prev Vet Med. 2020;184:1-6. doi: 10.1016/j.prevetmed.2020.105159.
https://doi.org/10.1016/j.prevetmed.2020... . 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 ²2. Jansen AM, Xavier SCDC, Roque ALR. Trypanosoma cruzi transmission in the wild and its most important reservoir hosts in Brazil. Parasit Vectors. 2018;11(1):1-25. doi: 10.1186/s13071-018-3067-2.
https://doi.org/10.1186/s13071-018-3067-... . Some etiological agents of these zoonoses, such as trypanosomatids, including Trypanosoma and Leishmania (Euglenozoa: Kinetoplastea, Trypanosomatidae), are important in veterinary and human medicine ¹1. Jaimes-Dueñez J, Cantillo-Barraza O, Triana-Chavez O, Mejia-Jaramillo AM. Molecular surveillance reveals bats from eastern Colombia infected with Trypanosoma theileri and Trypanosoma wauwau-like parasites. Prev Vet Med. 2020;184:1-6. doi: 10.1016/j.prevetmed.2020.105159.
https://doi.org/10.1016/j.prevetmed.2020... ^,33. Amórtegui-Hernández DM. (2022) Evaluación de la diversidad de parásitos del orden Trypanosomatida que infectan murciélagos en diferentes ecorregiones de Colombia [tesis maestría]. Facultad de Ciencias Biológicas, Universidad de los Andes (Bogotá D.C., Colombia); 2022. Disponible en: http://hdl.handle.net/1992/59458.
http://hdl.handle.net/1992/59458... .

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 ⁴4. Instituto Nacional de Salud-INS. Boletín Epidemiológico Semanal. Semana 50 (del 10 al 16 de diciembre) [Internet]. Instituto Nacional de Salud; 2023 [consultado el 28 de diciembre de 2023]. Disponible en: https://www.ins.gov.co/buscador-eventos/BoletinEpidemiologico/2023_Bolet%C3%ADn_epidemiologico_semana_50.pdf.
https://www.ins.gov.co/buscador-eventos/... . The way this disease spreads (transmission cycles) changes depending on the animals living in each area (ecotope). This shift is seen with T. cruziand 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 ⁵5. de Fuentes-Vicente JA, Gutiérrez-Cabrera AE, Flores-Villegas AL, Lowenberger C, Benelli G. What makes an effective Chagas disease vector? Factors underlying Trypanosoma cruzi-triatomine interactions. Acta Trop. 2018;183:23-31. doi: 10.1016/j.actatropica.2018.04.008.
https://doi.org/10.1016/j.actatropica.20... .

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 marinkelleiin 25 samples, and other Schizotrypanum species in 315 samples ⁶6. Marinkelle CJ. Prevalence of Trypanosoma cruzi-like infection of Colombia bats. Ann Trop Med Parasitol. 1982;76:125-134. doi: 10.1080/00034983.1982.11687517.
https://doi.org/10.1080/00034983.1982.11... . 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.

THE STUDY

Study area

The sampling area consisted of four collection points, two in urban ecotopes delimited as point 1U (11º01'02" N-74º52'30" W) and point 2U (11º01'00" N-74º52'28" W), within 895.6 ha of tropical dry forest on the Campus of the Universidad del Atlántico in the Puerto Colombia Municipality (average elevation: 21 meters), with an average temperature between 28°-32° C and an annual rainfall of 819 mm³, and two in wild ecotopes delimited as point 1W (10º47'54" N - 75º00'35" W) and point 2W (10º47'53" N - 75º00'34" W) formed within 1,425.2 ha of tropical dry forest in the Corregimiento of Chorrera (Juan de Acosta Municipality) (average elevation: 70 meters), average temperature between 27°-32° C and annual rainfall of 1,655 mm³, all from the Atlántico Department, northern of Colombia ⁷7. Instituto Von Humboldt. Actualización del mapa nacional Bosque seco tropical a escala de 1: 100.000. Atlántico-Colombia [Internet]; 2022 [consultado el 15 de junio de 2022]. Disponible en: https://www.humboldt.org.co/en/research/projects/developing-projects/item/158-bosques-secos-tropicales-en-colombia.
https://www.humboldt.org.co/en/research/... (Figure 1).

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) ⁸8. Bracamonte JC. Protocolo de muestreo para la estimación de la diversidad de murciélagos con redes de niebla en estudios de ecología. Ecol Austral. 2018;28(1):446-454. doi: 10.25260/EA.18.28.2.0.272.
https://doi.org/10.25260/EA.18.28.2.0.27... . The total effort was 4,320 m2 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 Díazet al.⁹9. Díaz MM, Solari S, Gregorin R, Aguirre LF, Barquez RM. Claves de identificación de los murciélagos Neotropicales: Chave de Identificação dos Morcegos Neotropicais. Tucumán: Argentina; 2021 Ed. PCMA: 10-132.. Sex, age, and reproductive condition were determined following methods in Kunzet al. ¹⁰10. Kunz TH, Wemmer C, Hayssen V. Sex, age and reproductive condition of mammals. Wilson DE, Cole R, Nichols J, Rudran R, Foster M (eds.). In: Measuring and monitoring biological diversity: standard methods for mammals. Washington: Smithsonian Institution Press; 1996: 279-290.. Microperforations were made in the left wing patagium to register recaptured. Another volume of blood was destined for parasitological analysis. The morphometric 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 ⁽¹¹11. Añez N, Crisante G, Soriano PJ. Trypanosoma cruzi congenital transmission in wild. Acta Trop. 2009; 109(1):78-80. doi: 10.1016/j.actatropica.2008.08.009.
https://doi.org/10.1016/j.actatropica.20... .

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) ¹²12. Noyes HA, Stevens JR, Teixeira M, Phelan J, Holz P. A nested PCR for the ssrRNA gene detects Trypanosoma binneyi in the platypus and Trypanosoma sp. in wombats and kangaroos in Australia. Int J Parasitol. 1999;29(1):331-339. doi: 10.1016/s0020-7519(98)00167-2.
https://doi.org/10.1016/s0020-7519(98)00... . 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 ¹²12. Noyes HA, Stevens JR, Teixeira M, Phelan J, Holz P. A nested PCR for the ssrRNA gene detects Trypanosoma binneyi in the platypus and Trypanosoma sp. in wombats and kangaroos in Australia. Int J Parasitol. 1999;29(1):331-339. doi: 10.1016/s0020-7519(98)00167-2.
https://doi.org/10.1016/s0020-7519(98)00... . To ensure the test's accuracy, we included a negative control (PCR mix without DNA) and a positive control (T. cruziDNA, strain MDID/CO/2018/Dm006).

The PCR mix was carried out with a final volume of 25 µL, of which 5 µL corresponded to the template DNA and the remaining 20 µL to 10 µL of GoTaq® Green Master Mix, 2X (Promega), 3 µL of MgCl2, 2 µL of each of the primers implemented for each stage and 5 μl of nuclease-free water (Promega®). PCR was carried out in the TC-9639 thermocycler (Benchmark SCIENTIFIC: Sayreville, NJ, USA) following the conditions of Noyes et al.¹²12. Noyes HA, Stevens JR, Teixeira M, Phelan J, Holz P. A nested PCR for the ssrRNA gene detects Trypanosoma binneyi in the platypus and Trypanosoma sp. in wombats and kangaroos in Australia. Int J Parasitol. 1999;29(1):331-339. doi: 10.1016/s0020-7519(98)00167-2.
https://doi.org/10.1016/s0020-7519(98)00... . 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 ¹²12. Noyes HA, Stevens JR, Teixeira M, Phelan J, Holz P. A nested PCR for the ssrRNA gene detects Trypanosoma binneyi in the platypus and Trypanosoma sp. in wombats and kangaroos in Australia. Int J Parasitol. 1999;29(1):331-339. doi: 10.1016/s0020-7519(98)00167-2.
https://doi.org/10.1016/s0020-7519(98)00... , 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 ¹³13. Hill MO. Diversity and evenness: a unifying notation and its consequences. Ecol. 1973;54(2):427-432. doi: 10.2307/1934352.. For this analysis, species rarefaction curves (Hill numbers) were performed using the "iNext" package in the R language ¹⁴14. Hsieh TC, Ma KH, Chao A. iNEXT: an R package for rarefaction and extrapolation of species diversity (Hill numbers). Methods Ecol Evol. 2016;7(12):1451-1456. doi: 10.1111/2041-210X.12613.
https://doi.org/10.1111/2041-210X.12613.... . 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.

FINDINGS

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 ¹⁵15. Gardner AL. Mammals of South America, Volume 1. Marsupials, Xenarthrans, Shrews, and Bats. The University of Chicago Press, Chicago and London; 2007.. The most abundant families wereMolossidae(62/125, 49.6%) andPhyllostomidae(43/125, 34.4%), followed byNoctilionidae(18/125, 14.4%) andVespertilionidae(2/125, 1.6%).

Within the general external morphological characteristics of the bats studied, individuals ofMolossus 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 ¹⁵15. Gardner AL. Mammals of South America, Volume 1. Marsupials, Xenarthrans, Shrews, and Bats. The University of Chicago Press, Chicago and London; 2007.. In addition,Noctilio albiventrisindividuals (Figure 2B) feature short, reddish-yellow fur, weighs 26.7 g, and have a forearm length of 59,3 mm and a wingspan of 421.6 mm ¹⁵15. Gardner AL. Mammals of South America, Volume 1. Marsupials, Xenarthrans, Shrews, and Bats. The University of Chicago Press, Chicago and London; 2007..

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 one), 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. molossusfrom the wild ecotope was found positive with 2.5% (3/118), while in the urban ecotopeN. albiventriswith 1.6% (2/118).

The frequency of infection byTrypanosomaspp. 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 wereM. molossus(for wild ecotopes) andN. albiventris(for urban ecotopes). The parasitological diagnosis forM. molossuswas 8.1% (5/61), regarding the molecular (4.9%, 3/61). N. albiventrispresented the same frequency of infection through parasitological and molecular diagnosis (16.6%, 2/12).

No association was found between the presence ofTrypanosomaspp. 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 ¹⁶16. Avendaño-Maldonado LJ, Camargo-Alarcón MA, Borja-Acuña R, Chacón-Pacheco J. Mamíferos del Departamento del Atlántico, Colombia. Biota Colombiana. 2021; 22(2):108-126. doi: 10.21068/c2021.v22n02a06.
https://doi.org/10.21068/c2021.v22n02a06... and 44.4% of those recorded in the Neotropics ¹⁷17. Oliveira da Silva AC. Infecção por tripanossomatídeos em quirópteros capturados no município de Natal-RN [tesis de maestría]. Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, 2021. Disponible en: https://repositorio.ufrn.br/handle/123456789/32416.
https://repositorio.ufrn.br/handle/12345... .

When looking at the total number of bat species (q0), our data suggests a higher potential species richness in the wild habitat 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 sufficient 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. ¹¹11. Añez N, Crisante G, Soriano PJ. Trypanosoma cruzi congenital transmission in wild. Acta Trop. 2009; 109(1):78-80. doi: 10.1016/j.actatropica.2008.08.009.
https://doi.org/10.1016/j.actatropica.20... , with evidence of congenital transmission of T. cruzi, and in Brazil by Oliveira da Silva ¹⁷17. Oliveira da Silva AC. Infecção por tripanossomatídeos em quirópteros capturados no município de Natal-RN [tesis de maestría]. Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, 2021. Disponible en: https://repositorio.ufrn.br/handle/123456789/32416.
https://repositorio.ufrn.br/handle/12345... 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 studied in the region of Vichada, with an infection frequency of 83.3% (10/12) for T. cruzi¹1. Jaimes-Dueñez J, Cantillo-Barraza O, Triana-Chavez O, Mejia-Jaramillo AM. Molecular surveillance reveals bats from eastern Colombia infected with Trypanosoma theileri and Trypanosoma wauwau-like parasites. Prev Vet Med. 2020;184:1-6. doi: 10.1016/j.prevetmed.2020.105159.
https://doi.org/10.1016/j.prevetmed.2020... , and in the region of Casanare, with the first report of L. amazonensis in this species, without data on infection frequency ³3. Amórtegui-Hernández DM. (2022) Evaluación de la diversidad de parásitos del orden Trypanosomatida que infectan murciélagos en diferentes ecorregiones de Colombia [tesis maestría]. Facultad de Ciencias Biológicas, Universidad de los Andes (Bogotá D.C., Colombia); 2022. Disponible en: http://hdl.handle.net/1992/59458.
http://hdl.handle.net/1992/59458... ; in turn, the same author analyzed blood samples from six bats from the Atlántico Department, without specifying the species, but noting the absence of trypanosomatids infections. Thus, the present study would be the first report of the species M. molossus as a host for Trypanosoma spp. in the Atlántico Department.

The presence of T. cruzi-TcBat in N. albiventris from Brazil was reported by Lima et al. ¹⁸18. Lima L, Espinosa-Álvarez O, Ortiz P, Trejo-Varón JA, Carranza JC, Pinto CM, et al. Genetic diversity of Trypanosoma cruzi in bats, and multilocus phylogenetic and phylogeographical analyses supporting Tcbat as an independent DTU (Discrete Typing Unit). Acta Trop. 2015;151(1):166-177. doi: 10.1016/j.actatropica.2015.07.015.
https://doi.org/10.1016/j.actatropica.20... . Marinkelle ⁶6. Marinkelle CJ. Prevalence of Trypanosoma cruzi-like infection of Colombia bats. Ann Trop Med Parasitol. 1982;76:125-134. doi: 10.1080/00034983.1982.11687517.
https://doi.org/10.1080/00034983.1982.11... reported infection with Schizotrypanum sp. in N. labialis, a synonym of N. leporinus, using parasitological techniques (50.0%, 157/315); this synonym was established as a species by Solari et al. ¹⁹19. Solari S, Muñoz-Saba Y, Rodríguez-Mahecha JV, Defler TR, Ramírez-Chaves HE, Trujillo F. Riqueza, endemismo y conservación de los mamíferos de Colombia. Mastozool neotrop [Internet] 2013;20(2):301-365. Disponible en: http://www.scielo.org.ar/scielo.php?script=sci_arttext&pid=S0327-93832013000200008&lng=es&nrm=iso.
http://www.scielo.org.ar/scielo.php?scri... . The present results would correspond to the first record of N. albiventris as a host of Trypanosoma spp. in Colombia. It was not possible to obtain the surrounding Trypanosoma species by genomic sequencing in the present study due to resource limitations.

In conclusion, the results suggest that the presence of bats infected with Trypanosoma spp. (with observed blood trypomastigotes) represents a potential risk, as they may come into contact with biological vectors, with the possibility of causing zoonotic diseases. Future studies will be necessary to identify the Trypanosoma species in bats and their role in the epidemiological scenario as a reservoir of trypanosomatids. This study contributes to strengthening the inventories of bat fauna in the municipalities of the Atlántico Department in the Caribbean region of Colombia.

References

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History