Detection of Trypanosomatid hemoflagellates in generalist bats from Costa Rica

Detection of Trypanosomatid hemoflagellates in generalist bats from Costa Rica | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Detection of Trypanosomatid hemoflagellates in generalist bats from Costa Rica Randall Rubí-Chacón, Andrea Urbina-Villalobos, Karen Daniela Sibaja-Morales, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7466101/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Hemoflagellates of the genera Leishmania and Trypanosoma have been reported in bats from various geographic regions worldwide. In Costa Rica, the presence of trypanosomatids in bats has been limited to descriptions of Trypanosoma vespertilionis and Trypanosoma leonidasdeanei in the 1950s and 1960s, with no prior reports of Leishmania spp. in these mammals. This study aimed to detect trypanosomatid hemoflagellates in generalist bat species from Costa Rica. Methods: Between June 2013 and August 2014, a total of 194 bats were captured from 11 localities across five provinces. Blood samples were collected from 98 individuals and analyzed by polymerase chain reaction (PCR) for the presence of Leishmania spp. and Trypanosoma spp. Results: Four samples (4.1%) tested positive for Leishmania spp. and nine samples (9.2%) for Trypanosoma spp. Sequencing determined for the first time the presence of Leishmania panamensis and Trypanosoma cruzi in generalist bats from Costa Rica, and confirmed the presence of Trypanosoma minasense in these volant mammals. Conclusions: These findings underscore the importance of integrative studies involving hosts, vectors, and environmental factors to understand the impact of ecosystem changes on trypanosomatid distribution and their potential implications for public health. It is necessary to understand the role that these hemoflagellates play on the health of bats, the importance of these mammals in leishmaniasis and trypanosomiasis, the transmission cycles and the ecological and bioclimate factors associated with vector and wildlife dynamics. Leishmania spp. Trypanosoma spp. wildlife polymerase chain reaction Background Bats are among the most successful groups of mammals and play key roles in the ecological dynamics of tropical forests ( 1 , 2 ). They exhibit remarkable biological diversity, including a wide variety of feeding strategies ( 3 ), and possess specialized morphological and physiological adaptations that enable them to fly ( 4 ). These traits have allowed bats to colonize a broad range of ecosystems, including both wild and domestic environments ( 5 ), demonstrating their adaptability and highlighting their potential importance as reservoirs or vectors of diverse pathogens ( 6 ). Over their evolutionary history, bats have coevolved with numerous pathogens ( 7 ). They have been associated with a wide array of infectious agents, including viruses ( 8 – 11 ), bacteria 12, 13), fungi ( 14 ), and parasitic protozoa such as trypanosomatids ( 15 , 16 ). Trypanosomatid hemoflagellates have been reported in bats from both Africa ( 17 ) and the Neotropics ( 15 , 19 – 21 ). In the Americas, several Leishmania species have been identified in bats, including Leishmania chagasi in Carollia perspicillata from Venezuela ( 18 ), and Leishmania amazonensis , Leishmania braziliensis , and Leishmania infantum in Brazilian bat species ( 19 – 21 ). The presence of Trypanosoma spp. in bats has been recognized since the early 20th century ( 22 ) and is a subject of interest in wildlife parasitology ( 23 ). Trypanosoma cruzi , the etiological agent of Chagas disease, has been reported in bats from Panama ( 23 , 24 ), the United States ( 15 ), Mexico ( 25 , 26 ), and Brazil ( 27 ). Additional reports include Trypanosoma evansi in Desmodus rotundus ( 28 ), and in nectarivorous bats ( Leptonycteris curasoae ) from Venezuela ( 29 ), and Trypanosoma cruzi marinkellei and Trypanosoma dionisii in Carollia perspicillata from Bolivia ( 30 ). As with Leishmania , the role of bats in the transmission cycles of Trypanosoma spp. remains unclear. In Costa Rica, reports of trypanosomatids in bats are limited to the identification of Trypanosoma vespertilionis in five Glossophaga mutica individuals from Santa Ana, San José province ( 31 ), and Trypanosoma leonidasdeanei in a Saccopteryx bilineata individual from Guápiles, Limón province ( 32 ). To date, no reports have confirmed the presence of Leishmania spp. or T. cruzi in Costa Rican bats. This study aimed to detect Leishmania spp. and T. cruzi in the blood of generalist bat species, given that Costa Rica is endemic for cutaneous leishmaniasis ( 33 ) and Chagas disease ( 34 ). Methods Study Area and Sampling Design Between June 2013 and August 2014, a descriptive, cross-sectional observational study was conducted using randomized sampling of bats in forest edges and woodland areas across 11 localities in different ecological life zones of Costa Rica. These regions ranged from very dry to very humid climates, with variable temperatures and precipitation patterns (35). The sampling sites included Talamanca (Kekoldi Reserve; 9°38′16″N, 82°47′47″W), Turrialba (Wagelia Reserve; 9°56′45″N, 83°41′24″W), Puerto Viejo de Sarapiquí (Pozo Azul Reserve, 10°23′56″N, 84°07′41″W; Finca Starke, 10°26′19″N, 84°00′02″W; and Ara Ambigua, 10°27′22″N, 84°01′32″W), Guápiles (Finca Corbana, 10°09′52″N, 83°46′33″W), Orosí (Finca Navarro, 9°49′33″N, 83°52′37″W), Tapantí National Park (9°46′52″N, 83°48′44″W), Carara National Park (9°46′48″N, 84°36′19″W), Santa Rosa National Park (10°50′04″N, 85°36′43″W), and Barra Honda National Park (10°11′14″N, 85°19′07″W). Bat Capture, Identification, and Blood Sampling Bats were captured using four Japanese mist nets (type BWF 50 denier/2 ply) with 1½-inch mesh openings and dimensions of 12 m × 2.5 m, set at ground level. All captured individuals were identified to species level based on external morphological characteristics (36, 37). Body weight, sex, reproductive status, and age group were recorded. Blood samples were collected via venipuncture of the propatagial or patagial vein using 27- or 30-gauge needles, following the protocol by Schinnerl et al. (38). After sampling, bats were evaluated for condition, given a glucose-rich solution orally, and released. Blood was collected in 1 mL EDTA-treated MiniCollect® tubes, stored at 4°C during transport, and then frozen at −20°C until analysis. DNA Extraction, Polymerase Chain Reaction (PCR) and Sequencing for Detection of Leishmania spp. and Trypanosoma spp. DNA was extracted from blood samples using the QIAamp® DNA Investigator Kit (QIAGEN, Crawley, UK), following the manufacturer’s protocol. Conventional PCR for Leishmania spp. was first performed using primers 13A (5′-GTG GGG GAG GGG CGT TCT-3′) and 13B (5′-ATT TTA CAC CAA CCC CCA GTT-3′), which amplified a 120 bp conserved region of kinetoplast DNA (kDNA) (39). Positive samples were subsequently analyzed with primers LITSR (5′-CTG GAT CAT TTT CCG ATG-3′) and L5.8S (5′-TGA TAC CAC TTA TCG CAC TT-3′) targeting a 330 bp ITS-1 region (40) for sequencing. Trypanosoma spp. detection was performed by nested PCR following the protocol described by Savani et al. (2005). The first round used primers S4 (5′-GAT CCA GCT GCA GGT TCA CC-3′) and S12 (5′-GGT TGA TTC CGT CCA CGG AC-3′), amplifying a 540 bp segment of the 18S rDNA gene. The second round used primers S17 (5′-CCA AGC TGC CCA GTA GAA T-3′) and S18 (5′-TCG GGC GGA TAA AAC ACC-3′), amplifying a 480 bp segment of the same gene. Positive controls included DNA from Leishmania panamensis , L. braziliensis , and L. chagasi strains, kindly provided by Dr. Azael Saldaña (Gorgas Memorial Institute, Panama), and DNA from T. cruzi isolated from Triatoma dimidiata. Nuclease-free water was used as a negative control. DNA amplicons were separated by electrophoresis on 2% agarose gels in 1× TBE buffer (Tris-Borate-EDTA, pH 8.0), stained with GelRed™ nucleic acid dye, and run at 90 V for 30 minutes. Bands were visualized under UV light using a BioDoc-It Imaging System. Band sizes were estimated using the GenRuler™ 100 bp DNA Ladder Plus (Fermentas®). Samples yielding amplicons of 120 bp and 330 bp were considered positive for Leishmania spp., while those with bands of 540 bp and 480 bp were considered positive for Trypanosoma spp. Positive PCR products were submitted for sequencing at MACROGEN® (South Korea). Sequences were edited using BIOEDIT 7.2.5 and manually refined. BLAST searches were conducted to compare sequences with those available in GenBank for species-level identification. Results A total of 194 generalist bats were captured across the 11 sampling localities, and blood samples were successfully obtained from 98 individuals. Most of these bats belonged to the genera Carollia, Artibeus , Sturnira , and Glossophaga . Conventional PCR based on kDNA amplification detected Leishmania spp. in four of the 98 blood samples analyzed, corresponding to a 4.1% infection rate. The Leishmania -positive bats belonged to the species Artibeus jamaicensis , Artibeus lituratus , Sturnira parvidens , and Carollia castanea . Table 1 summarizes the bat species, capture sites, and individual characteristics of these hosts. Table 1. Bat species, capture sites, and host characteristics for individuals PCR-positive for Leishmania spp. kDNA amplification Bat species Capture site Sex Weight (g) Age group Reproductive status Artibeus jamaicensis Barra Honda National Park Female 50 Adult Inactive Artibeus lituratus Këkoldi Reserve, Talamanca Female 34.5 Adult Active Sturnira parvidens Navarro-Orosí, Cartago Male 22 Adult Active Carollia castanea Wagelia, Turrialba, Cartago Female 13 Adult Inactive Only DNA from the male S. parvidens could be amplified in the ITS-1 PCR. Sequencing and BLAST analysis of the amplicon revealed that the sample had 98% identity with Leishmania panamensis (GenBank accession MT606233) isolated from ulcerated lesions in humans in Brazil. The sequence obtained in this study was deposited under GenBank accession PX060482. Nested PCR based on 18S rDNA amplification determined Trypanosoma spp. in nine out of 98 bat blood samples (9.2%). Sequencing and BLAST analysis of the 480 bp amplicons of the 18S rDNA gene revealed that three of the samples had 99–100% identity with T. cruzi , GenBank accession CP015657, isolated from the blood of a human in Brazil; corresponding sequences from this study are available under GenBank accessions PX090776, PX090777, and PX090778. Another three samples matched Trypanosoma minasense with 99–100% identity to GenBank AB362411, a sample isolated from a red-handed tamarin in Japan; these sequences were deposited under GenBank accessions PX090773, PX090774, and PX090775. The remaining three samples yielded amplicons consistent with Trypanosoma spp. but could not be identified at the species level based on BLAST results. Table 2 shows the species of bats, capture locations, identified trypanosomatid species, and host characteristics. Table 2. Bat species, capture sites, individual characteristics, and trypanosomatid species identified by 18S rDNA sequencing Bat species Capture site Identification 18S rDNA PCR Sex Weight (g) Age group Reproductive status Carollia perspicillata Finca Starke, Sarapiquí, Heredia T. cruzi Female 40 Adult Inactive Artibeus jamaicensis Pozo Azul, Sarapiquí, Heredia T. cruzi Male 49 Adult Inactive Artibeus jamaicensis Carara National Park, Puntarenas T. cruzi Female 48.5 Adult Inactive Glossophaga mutica Guápiles, Limón T. minasense Female 11 Adult Inactive Carollia sowelli Këkoldi Reserve, Talamanca, Limón T. minasense Female 18 Adult Active Glossophaga mutica Navarro-Oros i , Cartago T. minasense Male 12 Adult Inactive Carollia sowelli Carara National Park, Puntarenas Trypanosoma spp. Female 18 Adult Inactive Artibeus jamaicensis Ara Ambigua Farm, Sarapiquí, Heredia Trypanosoma spp. Female 48 Adult Active Carollia sowelli Pozo Azul, Sarapiquí, Heredia Trypanosoma spp. Male 20.5 Adult Inactive Discussion This study provides the first molecular evidence of Leishmania spp. in bats from Costa Rica, including the identification of L. panamensis , and confirms the presence of T. cruzi and T. minasense through 18S rDNA analysis, highlighting the potential role of generalist bats in the eco-epidemiology of trypanosomatid parasites in the Neotropics. The Leishmania infection rate found in bats in this study is comparable to the 3.7% reported in Brazil ( 19 ) but lower than 9.1% observed in Venezuela ( 18 ) and 8.9% reported in Mexico ( 41 ). Among the bat species found to harbor Leishmania spp. in this study, A. lituratus and S. parvidens have previously been associated with L. amazonensis in Brazil ( 19 ), while A. jamaicensis , A. lituratus , and S. parvidens were reported with L. mexicana in Mexico ( 41 , 42 ). The detection of Leishmania spp. DNA in C. castanea represents the first report of infection in this bat species. The geographic distribution of Leishmania -positive bats spanned four different localities in three provinces (Limón, Cartago, and Guanacaste). Positive bats were detected in endemic regions for Leishmania spp. (e.g., Turrialba-Cartago and Talamanca-Limón), where phlebotomine vectors have been reported ( 43 ), and in non-endemic areas (e.g., Orosi-Cartago and Barra Honda-Guanacaste), where bats were captured in caves—natural habitats for phlebotomine. These caves share climatic and ecological conditions with historically endemic regions, suggesting that bats in these areas may coexist with potential vectors of Leishmania . A bat of the species S. parvidens from Orosi was positive for L. panamensis . This parasite is the primary etiological agent of cutaneous leishmaniasis in the Caribbean region of Costa Rica and is recognized as the leading cause of this disease in the country ( 44 ). Notably, most of the Leishmania -positive bats were females. This may be explained by behavioral and ecological factors: males tend to fly higher and segregate, whereas females spend more time in roosts, caring for offspring, and flying at lower heights, increasing their likelihood of being captured with mist nets ( 18 ). Regarding the infection rate of Trypanosoma spp. detected in this study, this is comparable to the 10.2% prevalence reported in Panama ( 23 ), but lower than the 12.9% reported in Brazil ( 45 ), 36,5% in Ecuador ( 46 ), and 61,2% in Colombia ( 47 ). Conversely, it is higher than those 1,6% reported in Mexico ( 41 ) and 2,6% in Texas, USA ( 15 ). These variations in prevalence could be attributed to differences in ecological conditions, host species, and whether sampling was conducted exclusively in endemic areas or across both endemic and non-endemic regions, as was the case in this study. This is the first study to report the presence of T. minasense and T. cruzi in bats from Costa Rica, and the first to confirm infection through molecular techniques (PCR and sequencing). Species found infected with T. cruzi included A. jamaicensis and C. perspicillata , which have previously been reported as hosts in Panama ( 22 – 24 ), as well as in Colombia, Brazil, and Ecuador ( 45 – 47 ). The detection of T. minasense in G. mutica and C. sowelli represents the first report of this trypanosome in these bat species. Trypanosoma minasense has previously been reported in other mammalian hosts, such as non-human primates, including Saimiri sciureus imported to Japan from South America ( 48 ) and in all four species of monkeys from Costa Rica, living in different provinces in captivity as in the wild ( 49 ). Although its vector is still unknown, T. minasense is currently considered non-pathogenic for bats ( 49 ). The distribution of Trypanosoma -positive bats extended across both Pacific and Caribbean coasts. Two T. cruzi -positive individuals were captured in Puerto Viejo, Sarapiquí, an area where T. dimidiata , the vector of Chagas disease, has been reported ( 50 , 51 ). Another T. cruzi -positive bat was found in Carara National Park, where T. dimidiata has not yet been documented but where environmental conditions, such as altitude, temperature, and precipitation, are consistent with its known range ( 49 ). Moreover, T. dimidiata is known to inhabit ecotopes such as bat caves, rock piles, and tree hollows ( 52 ), which coincide with the roosting habits of the bat species captured in this study ( 37 ). These findings warrant further investigation into the potential presence of T. dimidiata in areas where T. cruzi -positive bats were detected. T. minasense was exclusively detected on the Caribbean coast, in Orosi, Cartago province, and in Këkoldi (Talamanca), and Corbana (Guápiles), Limón province. Previous reports found T. minasense in non-human primates from four provinces (Puntarenas, Alajuela, Limón, and Heredia) in Costa Rica ( 53 ). Capture sites for Trypanosoma - and Leishmania -positive bats were typically characterized by fragmented habitats, agricultural fields, disturbed forests, and urban areas like those reported by Cottontail et al. 2009 ( 23 ) and Víquez-Rodríguez in 2015 ( 41 ), who observed higher trypanosomatid infection rates in altered landscapes. Such environments are associated with high densities of generalist bat species and reduced overall bat diversity ( 54 ), which could promote immunosuppression and increase contact with vectors, thereby enhancing parasite transmission ( 23 ). The impact of these hemoflagellates on the health of bats should be investigated, as well as their role in the maintenance of the parasite's life cycle. Habitat loss and extreme bioclimate events are causing changes in the reproductive vector cycles and their geographic distribution ( 55 ). Considering growing anthropogenic pressure on tropical ecosystems and the complex ecological interactions involved in Leishmania and Trypanosoma transmission cycles, it is crucial to conduct integrative studies on hosts, vectors, and environmental variables. Such approaches will improve our understanding of how ecosystem changes influence hemoflagellate circulation in bats and their potential implications for public health and disease emergence. Conclusions This study provides the first molecular evidence of Leishmania spp., including L. panamensis , as well as T. cruzi and T. minasense infections in bats from Costa Rica, underscoring the importance of these mammals as potential reservoirs or sentinels in the eco-epidemiology of trypanosomatid parasites. The detection of these parasites in both endemic and non-endemic areas, particularly within altered habitats, highlights the role of environmental and ecological factors—such as habitat fragmentation, bat behavior, and possible vector presence—in shaping transmission dynamics. These findings contribute to a better understanding of the distribution of trypanosomatid infections in the Neotropics and emphasize the need for integrated surveillance approaches that include the role of these mammals in disease transmission cycles and the impact of these parasites on the health of bats. Declarations Ethics and Animal Welfare All procedures for the capture and sampling of bats in this study were approved by the Bioethics Committee of the School of Veterinary Medicine, Universidad Nacional, Costa Rica. The study was conducted under the corresponding permits granted by CONAGEBIO (National Commission for Biodiversity Management, File No. 163) and SINAC (National System of Conservation Areas). Availability of data and materials The datasets used and analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors have no relevant financial or non-financial interests to disclose. Funding This work was supported by Fondo Institucional de Desarrollo Académico (FIDA), Universidad Nacional, Project 0021-12: Murciélagos como bioindicadores: Presencia de agentes infecciosos (virus, hongos y parásitos) en murciélagos en Costa Rica Author Contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by RR, AUV, KSM, MJZM, and GD. The first draft of the manuscript was written by RR, AUV, and GD, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. References Medellín RA, Gaona O. Seed dispersal by bats and birds in forest and disturbed habitats in Chiapas, México. Biotropica. 1999;31:432-41. Meyer CF, Fründ J, Lizano WP, Kalko EK. Ecological correlates of vulnerability to fragmentation in Neotropical bats. J Appl Ecol. 2008;45(1):381-91. Serra-Cobo J, Lopez-Roig M. Bats and emerging infections: an ecological and virological puzzle. In: Emerging and Re-emerging Viral Infections. Cham: Springer; 2016. p. 35–48. Rangel DA, Lisboa CV, Novaes RLM, Silva BA, Souza RDF, Jansen AM, et al. Isolation and characterization of trypanosomatids, including Crithidia mellificae, in bats from the Atlantic Forest of Rio de Janeiro, Brazil. PLoS Negl Trop Dis. 2019;13(7):e0007527. Rotureau B. Ecology of the Leishmania species in the Guianan ecoregion complex. Am J Trop Med Hyg. 2006;74(1):81–6. https://doi.org/10.4269/ajtmh.2006.74.81 Jansen AM, Roque ALR. Domestic and Wild Mammalian Reservoirs. In: American Trypanosomiasis. 2010;249–76. https://doi.org/10.1016/B978-0-12-384876-5.00011-333 Shipley R, Wright E, Selden D, Wu G, Aegerter J, Fooks AR, et al. Bats and viruses: emergence of novel lyssaviruses and association of bats with viral zoonoses in the EU. Trop Med Infect Dis. 2019;4(1):31. Schneider M, Burgoa C. Algunas consideraciones sobre la rabia humana transmitida por murciélago. Salud Publica Mex. 1995;37(4):354–62. Wang LF, Shi Z, Zhang S, Field H, Daszak P, Eaton BT. Review of bats and SARS. Emerg Infect Dis. 2006;12:1834–40. Calisher CH, Holmes KV, Dominguez SR, Schountz T, Cryan P. Bats prove to be rich reservoirs for emerging viruses. Microbe. 2008;3:521-8. Hui-Ju H, Hong-Ling W, Chuan-Min Z, Fang-Fang C, Li-Mei L, Jian-Wei L, et al. Los murciélagos como reservorios de enfermedades infecciosas emergentes graves. Virus Res. 2015;205:1-6. https://doi.org/10.1016/j.virusres.2015.05.006 Mühldorfer K. Bats and bacterial pathogens: a review. Zoonoses Public Health. 2013;60(1):93-103. Torres-Castro M, Febles-Solís V, Hernández-Betancourt S, Noh-Pech H, Estrella E, Peláez-Sánchez R, et al. Leptospira patógenas en murciélagos de Campeche y Yucatán, México. Rev MVZ Córdoba. 2020;25:e1815. https://doi.org/10.21897/rmvz.1815 Panizo MM, Dolande M, Reviakina V, Maldonado B. Histoplasmosis pulmonar asociada con visita a cuevas: Descripción de un brote epidémico y revisión de la literatura. Rev Soc Venez Microbiol. 2001;21(1):30-5. Hodo CL, Goodwin CC, Mayes BC, Mariscal JA, Waldrup KA, Hamer SA. Trypanosome species, including Trypanosoma cruzi , in sylvatic and peridomestic bats of Texas, USA. Acta Trop. 2016;164:259-66. Jansen AM, das Chagas Xavier SC, Roque ALR. Trypanosoma cruzi transmission in the wild and its most important reservoir hosts in Brazil. Parasites Vectors. 2018;11(1):1-25. Kassahun A, Sadlova J, Benda P, Kostalova T, Warburg A, Hailu A, et al. Natural infection of bats with Leishmania in Ethiopia. Acta Trop. 2015. http://dx.doi.org/10.1016/j.actatropica.2015.07.024 Lima HD, Rodríguez N, Barrios M, Ávila Á, Cañizales I, Gutiérrez S. Isolation and molecular identification of Leishmania chagasi from a bat (Carollia perspicillata) in northeastern Venezuela. Mem Inst Oswaldo Cruz. 2008;103:412-4. Savani ES, de Almeida MF, De Oliveira Camargo MC, D’Auria SR, Silva MM, de Oliveira ML, et al. Detection of Leishmania amazonensis and Leishmania infantum chagasi in Brazilian bats. Vet Parasitol. 2010;168:5–10. Shapiro JT, da Costa Lima Junior MS, Dorval MEC, de Oliveira França A, Cepa Matos MDF, Bordignon MO. First record of Leishmania braziliensis presence detected in bats, Mato Grosso do Sul, southwest Brazil. Acta Trop. 2013;128:171–4. Castro LS, Dorval ME, Matheus LM, Bednaski AV, Facco GG, Silveira M, et al. Leishmania presence in bats in areas endemic for leishmaniasis in central-west Brazil. Int J Parasitol Parasites Wildl. 2020;11:261-7. Hoare CA. The Trypanosomes of Mammals. A Zoological Monograph. Oxford and Edinburgh: Blackwell Scientific Publications; 1972. Cottontail VM, Wellinghausen N, Kalko EKV. Habitat fragmentation and haemoparasites in the common fruit bats Artibeus jamaicensis (Phyllostomidae) in tropical lowland forest in Panamá. Parasitology. 2009;136(10):1133-45. Cottontail VM, Kalko EK, Cottontail I, Wellinghausen N, Tschapka M, Perkins SL, et al. High local diversity of Trypanosoma in a common bat species, and implications for the biogeography and taxonomy of the T. cruzi clade. PLoS One. 2014;9:e108603. Torres-Castro M, Cuevas-Koh N, Hernández-Betancourt S, Noh-Pech H, Estrella E, Herrera-Flores B, et al. Natural infection with Trypanosoma cruzi in bats captured in Campeche and Yucatán, México. Biomédica. 2021;41(Supl.1):131–40. https://doi.org/10.7705/biomedica.5450 Moo-Millan JI, Tu W, Montalvo-Balam TJ, Ibarra-López MP, Hernández-Betancourt S, May-Concha IJ, et al. Presence of Trypanosoma cruzi TcI and Trypanosoma dionisii in sylvatic bats from Yucatan, Mexico. Trans R Soc Trop Med Hyg. 2024;trae023. França DA, Louro M, Zúquete S, Zanini DS, Moraes GN, Rocha GS, et al. Trypanosoma cruzi in Bats (Chiroptera; Mammalia) from the Brazilian Atlantic Forest, São Paulo State. Microorganisms. 2024;12:945. https://doi.org/10.3390/microorganisms12050945 Brun R, Hecker H, Lun ZR. Trypanosoma evansi and T. equiperdum : distribution, biology, treatment and phylogenetic relationship (a review). Vet Parasitol. 1998;79(2):95-107. Silva-Iturriza A, Nassar JM, Garcia-Rawlins AM, Rosales R, Mijares A. Trypanosoma evansi kDNA minicircle found in the Venezuelan nectar-feeding bat Leptonycteris curasoae (Glossophaginae), supports the hypothesis of multiple origins of that parasite in South America. Parasitol Int. 2013;62(2):95–9. Torrico MC, Téllez T, Tenorio O, Rojas L, Huaranca JC, De la Barra A, et al. Tripanosomátidos aislados de mamíferos silvestres en tres departamentos de Bolivia (Cochabamba, Potosí y Santa Cruz de la Sierra). Gac Med Boliv. 2013;36(1):6–10. Zeledón R, Vieto PL. Hallazgo de Schizotrypanum vespertilionis (Battaglia, 1904) en la sangre de murciélagos de Costa Rica. Rev Biol Trop. 1957;5:123–8. Zeledón R, Rosabal R. Trypanosoma leonidasdeanei sp. nov. in insectivorous bats of Costa Rica. Ann Trop Med Parasitol. 1969;63:221–8. Jaramillo-Antillón O, Espinoza-Aguirre A, Lobo-Philp R. Estado actual de la leishmaniosis en Costa Rica. Acta Med Costarric. 2009;51:158–64. Zeledón R, et al. Chagas disease in Costa Rica. Am J Trop Med Hyg. 1975;24(3):405–408. Müller E. Investigaciones en frutos y semillas de árboles individuales de cinco especies forestales de la Región Huetar Norte de Costa Rica, con especial consideración en el almacenamiento. Documento 51. COSEFORMA, Costa Rica; 1997. 237 p. Timm RM, Laval RK, Rodríguez B. Clave de Campo para los Murciélagos de Costa Rica. Brenesia. 1999;52:1–32. Laval RK, Rodríguez-Herrera B. Murciélagos de Costa Rica. Santo Domingo, Heredia, Costa Rica: Editorial INBio; 2002. 310 p. Schinnerl M, Aydinonat D, Schwarzenberger F, Voigt C. Hematological survey of common neotropical bat species from Costa Rica. J Zoo Wildl Med. 2011;42:382–91. Medeiros ACR, Rodríguez SS, Roselino AMF. Comparison of the specificity of PCR and the histopathological detection of Leishmania for the diagnosis of American cutaneous leishmaniasis. Braz J Med Biol Res. 2002;35(4):421-4. El Tai NO, El Fari M, Mauricio I, Miles MA, Oskam L, El Safi SH, Presber WH, Schönian G. Leishmania donovani : intraspecific polymorphisms of Sudanese isolates revealed by PCR-based analyses and DNA sequencing. Exp Parasitol. 2001 Jan;97(1):35-44. doi:10.1006/expr.2001.4592. PMID: 11207112. Víquez-Rodríguez. Prevalencia de Leishmania mexicana y Trypanosoma cruzi en los murciélagos Carollia sowelli y Sturnira lilium bajo dos condiciones distintas de perturbación antropogénica en la Selva Lacandona, Chiapas. [Tesis de Maestría]. México: Universidad Nacional Autónoma de México; 2015. Berzunza-Cruz M, Rodríguez-Moreno Á, Gutiérrez-Granados G, González-Salazar C, Stephens CR, Hidalgo-Mihart M, et al. Leishmania (L.) mexicana infected bats in Mexico: novel potential reservoirs. PLoS Negl Trop Dis. 2015;9(1):e0003438. Zeledón R, Murillo J, Gutiérrez H. Flebótomos antropófilos y leishmaniasis cutánea en Costa Rica. Bol Oficina Sanit Panam. 1985;99:163–72. Peraza J, Urbina A, Zeledón R. Zymodeme and serodeme characterization of Leishmania isolates obtained from Costa Rican patients. Mem Inst Oswaldo Cruz. 1998;93(3):283-7. Cavazzana M, Marcili A, Lima L, da Silva FM, Junqueira ÂC, Veludo HH, et al. Phylogeographical, ecological and biological patterns shown by nuclear (ssrRNA and gGAPDH) and mitochondrial (Cyt b) genes of trypanosomes of the subgenus Schizotrypanum parasitic in Brazilian bats. Int J Parasitol. 2010;40(3):345-55. Pinto CM, Ocaña-Mayorga S, Tapia EE, Lobos SE, Zurita AP, Aguirre-Villacís F, et al. Bats, trypanosomes, and triatomines in Ecuador: new insights into the diversity, transmission, and origins of Trypanosoma cruzi and Chagas disease. PLoS One. 2015;10:e0139999. http://dx.doi.org/10.1371/journal.pone.0139999 Ramírez JD, Tapia-Calle G, Muñoz-Cruz G, Poveda C, Rendón LM, Hincapié E, Guhl F. Trypanosoma species in neo-tropical bats: biological, evolutionary and epidemiological implications. Infect Genet Evol. 2014;22:250-6. Sato TM, Passos FC, Nogueira AC. Frugivoria de morcegos (Mammalia, Chiroptera) em Cecropia pachystachya (Urticaceae) e seus efeitos na germinação de sementes. Pap Avulsos Zool. 2008;48(3):19–26. Chinchilla M, Troyo A, Guerrero OM, Gutiérrez-Espeleta GA, Sánchez R. Presencia de Trypanosoma minasense (Kinetoplastida: Trypanosomatidae) en Alouatta palliata (Primates: Cebidae) de Costa Rica. Parasitol Latinoam. 2005;60(1-2):90-2. Cottontail VM, Kalko EKV, Cottontail I, Wellinghausen N, Tschapka M, et al. High local diversity of Trypanosoma in a common bat species, and implications for the biogeography and taxonomy of the T. cruzi clade. PLoS One. 2014;9(9):e108603. doi:10.1371/journal.pone.0108603. Zeledón R, Calvo N, Montenegro VM, Lorosa ES, Arévalo C. A survey on Triatoma dimidiata in an urban area of the province of Heredia, Costa Rica. Mem Inst Oswaldo Cruz. 2005;100(6):507-12. Zeledón R. Triatoma dimidiata y su relación con la enfermedad de Chagas . San José (CR): EUNED; 1981. 164 p. Solorzano-Morales A, Tien C, Alfaro S, Dolz G. Trypanosomatid infections in non-human primates of Costa Rica. In: IV Bienal WDA Latinoamérica; 2019 Jul 16–18; San José, Costa Rica. Fenton MB, Acharya L, Audet D, Hickey MBC, Merriman C, Obrist MK, Adkins B. Phyllostomid bats (Chiroptera: Phyllostomidae) as indicators of habitat disruption in the Neotropics. Biotropica. 1992;24(3):440-6 . França DA, Louro M, Zúquete S, Zanini DS, Moraes GN, Rocha GS, Biondo LM, Fornazari F, Menozzi BD, Fonseca IP, et al. Trypanosoma cruzi in bats (Chiroptera; Mammalia) from the Brazilian Atlantic Forest, São Paulo State. Microorganisms. 2024;12(5):945. doi:10.3390/microorganisms12050945. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7466101","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":510934176,"identity":"34d32429-4937-438b-a5bc-652b2cef60e5","order_by":0,"name":"Randall Rubí-Chacón","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFElEQVRIiWNgGAWjYJACCYYCCyDFw3CAAYQYGBgfENZiIIGihdmAaC0MUC1sEviUm7efPXjjB1CL+YzcgwcYau7I87f3Hqvm3WHHwN9+gPFzBaYWmTN5yZY9QC0yN/ISDjAce2Y448y5tNu8Z5IZJM4kMEueweaoHDMJHqAWCYkcgwOMDYcZN0jkmN3mbWNmYLjBwMbYgEUL/xszyT9IWuxBWop52+oZ5HFpASqQRrYlEaSFmbftMIMBTi1vjK1lDCR4JHjeJRxIOHY4ecaZM8aSc9uO8xieSWyWxOqwHMObbyps5CTYcw9/+FBz2La/vcfww9u2ajm544cPfsSiBQbAkcKQgCqCzVmjYBSMglEwCogBAMhcXXtI/DyQAAAAAElFTkSuQmCC","orcid":"","institution":"National University of Costa Rica","correspondingAuthor":true,"prefix":"","firstName":"Randall","middleName":"","lastName":"Rubí-Chacón","suffix":""},{"id":510934177,"identity":"57d61166-613c-455d-a9f1-1c395f888479","order_by":1,"name":"Andrea Urbina-Villalobos","email":"","orcid":"","institution":"National University of Costa Rica","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Urbina-Villalobos","suffix":""},{"id":510934178,"identity":"fee66da6-2911-4dc9-aa3f-84139d264ea8","order_by":2,"name":"Karen Daniela Sibaja-Morales","email":"","orcid":"","institution":"Universidad Estatal a Distancia (UNED), Cátedra de Salud","correspondingAuthor":false,"prefix":"","firstName":"Karen","middleName":"Daniela","lastName":"Sibaja-Morales","suffix":""},{"id":510934179,"identity":"1fdab85e-2d82-4559-98ca-ed4c50671004","order_by":3,"name":"María José Zuniga-Moya","email":"","orcid":"","institution":"National University of Costa Rica","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"José","lastName":"Zuniga-Moya","suffix":""},{"id":510934180,"identity":"be3dea01-ce87-47eb-9c35-48752e3fb4c0","order_by":4,"name":"Gaby Dolz","email":"","orcid":"","institution":"National University of Costa Rica","correspondingAuthor":false,"prefix":"","firstName":"Gaby","middleName":"","lastName":"Dolz","suffix":""}],"badges":[],"createdAt":"2025-08-26 20:53:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7466101/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7466101/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91797878,"identity":"aecc08ec-a1a0-49f8-8430-e0c4a332b5e6","added_by":"auto","created_at":"2025-09-21 17:01:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":661963,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7466101/v1/865165d0-870a-4b3a-b62e-54a21b45f26f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Detection of Trypanosomatid hemoflagellates in generalist bats from Costa Rica","fulltext":[{"header":"Background","content":"\u003cp\u003eBats are among the most successful groups of mammals and play key roles in the ecological dynamics of tropical forests (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). They exhibit remarkable biological diversity, including a wide variety of feeding strategies (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), and possess specialized morphological and physiological adaptations that enable them to fly (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). These traits have allowed bats to colonize a broad range of ecosystems, including both wild and domestic environments (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), demonstrating their adaptability and highlighting their potential importance as reservoirs or vectors of diverse pathogens (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOver their evolutionary history, bats have coevolved with numerous pathogens (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). They have been associated with a wide array of infectious agents, including viruses (\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), bacteria 12, 13), fungi (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), and parasitic protozoa such as trypanosomatids (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTrypanosomatid hemoflagellates have been reported in bats from both Africa (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) and the Neotropics (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). In the Americas, several \u003cem\u003eLeishmania\u003c/em\u003e species have been identified in bats, including \u003cem\u003eLeishmania chagasi\u003c/em\u003e in \u003cem\u003eCarollia perspicillata\u003c/em\u003e from Venezuela (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), and \u003cem\u003eLeishmania amazonensis\u003c/em\u003e, \u003cem\u003eLeishmania braziliensis\u003c/em\u003e, and \u003cem\u003eLeishmania infantum\u003c/em\u003e in Brazilian bat species (\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe presence of \u003cem\u003eTrypanosoma\u003c/em\u003e spp. in bats has been recognized since the early 20th century (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) and is a subject of interest in wildlife parasitology (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e, the etiological agent of Chagas disease, has been reported in bats from Panama (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), the United States (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), Mexico (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), and Brazil (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Additional reports include \u003cem\u003eTrypanosoma evansi\u003c/em\u003e in \u003cem\u003eDesmodus rotundus\u003c/em\u003e (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), and in nectarivorous bats (\u003cem\u003eLeptonycteris curasoae\u003c/em\u003e) from Venezuela (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), and \u003cem\u003eTrypanosoma cruzi marinkellei\u003c/em\u003e and \u003cem\u003eTrypanosoma dionisii\u003c/em\u003e in \u003cem\u003eCarollia perspicillata\u003c/em\u003e from Bolivia (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). As with \u003cem\u003eLeishmania\u003c/em\u003e, the role of bats in the transmission cycles of \u003cem\u003eTrypanosoma\u003c/em\u003e spp. remains unclear.\u003c/p\u003e\u003cp\u003eIn Costa Rica, reports of trypanosomatids in bats are limited to the identification of \u003cem\u003eTrypanosoma vespertilionis\u003c/em\u003e in five \u003cem\u003eGlossophaga mutica\u003c/em\u003e individuals from Santa Ana, San Jos\u0026eacute; province (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), and \u003cem\u003eTrypanosoma leonidasdeanei\u003c/em\u003e in a \u003cem\u003eSaccopteryx bilineata\u003c/em\u003e individual from Gu\u0026aacute;piles, Lim\u0026oacute;n province (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). To date, no reports have confirmed the presence of \u003cem\u003eLeishmania\u003c/em\u003e spp. or \u003cem\u003eT. cruzi\u003c/em\u003e in Costa Rican bats.\u003c/p\u003e\u003cp\u003eThis study aimed to detect \u003cem\u003eLeishmania\u003c/em\u003e spp. and \u003cem\u003eT. cruzi\u003c/em\u003e in the blood of generalist bat species, given that Costa Rica is endemic for cutaneous leishmaniasis (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e) and Chagas disease (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e"},{"header":"Methods","content":"\u003ch4\u003eStudy Area and Sampling Design\u003c/h4\u003e\n\u003cp\u003eBetween June 2013 and August 2014, a descriptive, cross-sectional observational study was conducted using randomized sampling of bats in forest edges and woodland areas across 11 localities in different ecological life zones of Costa Rica. These regions ranged from very dry to very humid climates, with variable temperatures and precipitation patterns (35). The sampling sites included Talamanca (Kekoldi Reserve; 9\u0026deg;38\u0026prime;16\u0026Prime;N, 82\u0026deg;47\u0026prime;47\u0026Prime;W),\u003cbr\u003e\u0026nbsp;Turrialba (Wagelia Reserve; 9\u0026deg;56\u0026prime;45\u0026Prime;N, 83\u0026deg;41\u0026prime;24\u0026Prime;W), Puerto Viejo de Sarapiqu\u0026iacute; (Pozo Azul Reserve, 10\u0026deg;23\u0026prime;56\u0026Prime;N, 84\u0026deg;07\u0026prime;41\u0026Prime;W; Finca Starke, 10\u0026deg;26\u0026prime;19\u0026Prime;N, 84\u0026deg;00\u0026prime;02\u0026Prime;W; and Ara Ambigua, 10\u0026deg;27\u0026prime;22\u0026Prime;N, 84\u0026deg;01\u0026prime;32\u0026Prime;W), Gu\u0026aacute;piles (Finca Corbana, 10\u0026deg;09\u0026prime;52\u0026Prime;N, 83\u0026deg;46\u0026prime;33\u0026Prime;W),\u003cbr\u003e\u0026nbsp;Oros\u0026iacute; (Finca Navarro, 9\u0026deg;49\u0026prime;33\u0026Prime;N, 83\u0026deg;52\u0026prime;37\u0026Prime;W), Tapant\u0026iacute; National Park (9\u0026deg;46\u0026prime;52\u0026Prime;N, 83\u0026deg;48\u0026prime;44\u0026Prime;W), Carara National Park (9\u0026deg;46\u0026prime;48\u0026Prime;N, 84\u0026deg;36\u0026prime;19\u0026Prime;W), Santa Rosa National Park (10\u0026deg;50\u0026prime;04\u0026Prime;N, 85\u0026deg;36\u0026prime;43\u0026Prime;W), and Barra Honda National Park (10\u0026deg;11\u0026prime;14\u0026Prime;N, 85\u0026deg;19\u0026prime;07\u0026Prime;W).\u003cbr\u003e\u0026nbsp;\u003cstrong\u003eBat Capture, Identification, and Blood Sampling\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBats were captured using four Japanese mist nets (type BWF 50 denier/2 ply) with 1\u0026frac12;-inch mesh openings and dimensions of 12 m \u0026times; 2.5 m, set at ground level. All captured individuals were identified to species level based on external morphological characteristics (36, 37). Body weight, sex, reproductive status, and age group were recorded. Blood samples were collected via venipuncture of the propatagial or patagial vein using 27- or 30-gauge needles, following the protocol by Schinnerl et al. (38). After sampling, bats were evaluated for condition, given a glucose-rich solution orally, and released. Blood was collected in 1 mL EDTA-treated MiniCollect\u0026reg; tubes, stored at 4\u0026deg;C during transport, and then frozen at \u0026minus;20\u0026deg;C until analysis.\u003c/p\u003e\n\u003ch4\u003eDNA Extraction, Polymerase Chain Reaction (PCR) and Sequencing for Detection of\u0026nbsp;\u003cem\u003eLeishmania\u003c/em\u003e spp. and\u0026nbsp;\u003cem\u003eTrypanosoma\u003c/em\u003e spp.\u003c/h4\u003e\n\u003cp\u003eDNA was extracted from blood samples using the QIAamp\u0026reg; DNA Investigator Kit (QIAGEN, Crawley, UK), following the manufacturer\u0026rsquo;s protocol. Conventional PCR for\u0026nbsp;\u003cem\u003eLeishmania\u0026nbsp;\u003c/em\u003espp. was first performed using primers 13A (5\u0026prime;-GTG GGG GAG GGG CGT TCT-3\u0026prime;) and 13B (5\u0026prime;-ATT TTA CAC CAA CCC CCA GTT-3\u0026prime;), which amplified a 120 bp conserved region of kinetoplast DNA (kDNA) (39). Positive samples were subsequently analyzed with primers LITSR (5\u0026prime;-CTG GAT CAT TTT CCG ATG-3\u0026prime;) and L5.8S (5\u0026prime;-TGA TAC CAC TTA TCG CAC TT-3\u0026prime;) targeting a 330 bp ITS-1 region (40) for sequencing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTrypanosoma\u003c/em\u003e spp. detection was performed by nested PCR following the protocol described by Savani et al. (2005). The first round used primers S4 (5\u0026prime;-GAT CCA GCT GCA GGT TCA CC-3\u0026prime;) and S12 (5\u0026prime;-GGT TGA TTC CGT CCA CGG AC-3\u0026prime;), amplifying a 540 bp segment of the 18S rDNA gene. The second round used primers S17 (5\u0026prime;-CCA AGC TGC CCA GTA GAA T-3\u0026prime;) and S18 (5\u0026prime;-TCG GGC GGA TAA AAC ACC-3\u0026prime;), amplifying a 480 bp segment of the same gene. Positive controls included DNA from\u0026nbsp;\u003cem\u003eLeishmania panamensis\u003c/em\u003e,\u0026nbsp;\u003cem\u003eL. braziliensis\u003c/em\u003e, and\u0026nbsp;\u003cem\u003eL. chagasi\u003c/em\u003e strains, kindly provided by Dr. Azael Salda\u0026ntilde;a (Gorgas Memorial Institute, Panama), and DNA from\u0026nbsp;\u003cem\u003eT. cruzi\u003c/em\u003e isolated from\u0026nbsp;\u003cem\u003eTriatoma dimidiata.\u003c/em\u003e Nuclease-free water was used as a negative control.\u003c/p\u003e\n\u003cp\u003eDNA amplicons were separated by electrophoresis on 2% agarose gels in 1\u0026times; TBE buffer (Tris-Borate-EDTA, pH 8.0), stained with GelRed\u0026trade; nucleic acid dye, and run at 90 V for 30 minutes. Bands were visualized under UV light using a BioDoc-It Imaging System. Band sizes were estimated using the GenRuler\u0026trade; 100 bp DNA Ladder Plus (Fermentas\u0026reg;). Samples yielding amplicons of 120 bp and 330 bp were considered positive for\u0026nbsp;\u003cem\u003eLeishmania\u003c/em\u003e spp., while those with bands of 540 bp and 480 bp were considered positive for\u0026nbsp;\u003cem\u003eTrypanosoma\u003c/em\u003e spp. Positive PCR products were submitted for sequencing at MACROGEN\u0026reg; (South Korea). Sequences were edited using BIOEDIT 7.2.5 and manually refined. BLAST searches were conducted to compare sequences with those available in GenBank for species-level identification.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 194 generalist bats were captured across the 11 sampling localities, and blood samples were successfully obtained from 98 individuals. Most of these bats belonged to the genera \u003cem\u003eCarollia,\u003c/em\u003e \u003cem\u003eArtibeus\u003c/em\u003e, \u003cem\u003eSturnira\u003c/em\u003e, and \u003cem\u003eGlossophaga\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eConventional PCR based on kDNA amplification detected \u003cem\u003eLeishmania\u003c/em\u003e spp. in four of the 98 blood samples analyzed, corresponding to a 4.1% infection rate. The \u003cem\u003eLeishmania\u003c/em\u003e-positive bats belonged to the species \u003cem\u003eArtibeus jamaicensis\u003c/em\u003e, \u003cem\u003eArtibeus lituratus\u003c/em\u003e, \u003cem\u003eSturnira parvidens\u003c/em\u003e, and \u003cem\u003eCarollia castanea\u003c/em\u003e. Table 1 summarizes the bat species, capture sites, and individual characteristics of these hosts.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 1. Bat species, capture sites, and host characteristics for individuals PCR-positive for \u003cem\u003eLeishmania\u0026nbsp;\u003c/em\u003espp. kDNA amplification\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eBat species\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eCapture site\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eSex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eWeight (g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eAge group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003eReproductive status\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cem\u003eArtibeus jamaicensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eBarra Honda National Park\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003eInactive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cem\u003eArtibeus lituratus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eK\u0026euml;koldi Reserve, Talamanca\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e34.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003eActive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cem\u003eSturnira parvidens\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eNavarro-Oros\u0026iacute;, Cartago\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003eActive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cem\u003eCarollia castanea\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eWagelia, Turrialba, Cartago\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003eInactive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eOnly DNA from the male \u003cem\u003eS. parvidens\u0026nbsp;\u003c/em\u003ecould be amplified in the ITS-1 PCR. Sequencing and BLAST analysis of the amplicon revealed that the sample had 98% identity with \u003cem\u003eLeishmania panamensis\u003c/em\u003e (GenBank accession MT606233) isolated from ulcerated lesions in humans in Brazil. The sequence obtained in this study was deposited under GenBank accession PX060482.\u003c/p\u003e\n\u003cp\u003eNested PCR based on 18S rDNA amplification determined \u003cem\u003eTrypanosoma\u003c/em\u003e spp. in nine out of 98 bat blood samples (9.2%). Sequencing and BLAST analysis of the 480 bp amplicons of the 18S rDNA gene revealed that three of the samples had 99\u0026ndash;100% identity with \u003cem\u003eT. cruzi\u003c/em\u003e, GenBank accession CP015657, isolated from the blood of a human in Brazil; corresponding sequences from this study are available under GenBank accessions PX090776, PX090777, and PX090778. Another three samples matched \u003cem\u003eTrypanosoma minasense\u003c/em\u003e with 99\u0026ndash;100% identity to GenBank AB362411, a sample isolated from a red-handed tamarin in Japan; these sequences were deposited under GenBank accessions PX090773, PX090774, and PX090775. The remaining three samples yielded amplicons consistent with \u003cem\u003eTrypanosoma\u003c/em\u003e spp. but could not be identified at the species level based on BLAST results. Table 2 shows the species of bats, capture locations, identified trypanosomatid species, and host characteristics.\u003c/p\u003e\n\u003cp\u003eTable 2. Bat species, capture sites, individual characteristics, and trypanosomatid species identified by 18S rDNA sequencing\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eBat species\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003eCapture site\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003eIdentification 18S rDNA PCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eSex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eWeight (g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003eAge group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003eReproductive status\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e\u003cem\u003eCarollia perspicillata\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003eFinca Starke, Sarapiqu\u0026iacute;, Heredia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cem\u003eT. cruzi\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003eInactive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e\u003cem\u003eArtibeus jamaicensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003ePozo Azul, Sarapiqu\u0026iacute;, Heredia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cem\u003eT. cruzi\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003eInactive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e\u003cem\u003eArtibeus jamaicensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003eCarara National Park, Puntarenas\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cem\u003eT. cruzi\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e48.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003eInactive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e\u003cem\u003eGlossophaga mutica\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003eGu\u0026aacute;piles, Lim\u0026oacute;n\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cem\u003eT. minasense\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003eInactive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e\u003cem\u003eCarollia sowelli\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003eK\u0026euml;koldi Reserve, Talamanca, Lim\u0026oacute;n\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cem\u003eT. minasense\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003eActive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e\u003cem\u003eGlossophaga mutica\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003eNavarro-Oros\u003cins cite=\"mailto:Ricardo%20Barahona%20García\" datetime=\"2025-08-18T20:09\"\u003ei\u003c/ins\u003e, Cartago\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cem\u003eT. minasense\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003eInactive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e\u003cem\u003eCarollia sowelli\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003eCarara National Park, Puntarenas\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cem\u003eTrypanosoma\u0026nbsp;\u003c/em\u003espp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003eInactive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e\u003cem\u003eArtibeus jamaicensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003eAra Ambigua Farm, Sarapiqu\u0026iacute;, Heredia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cem\u003eTrypanosoma\u0026nbsp;\u003c/em\u003espp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003eActive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eCarollia sowelli\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003ePozo Azul, Sarapiqu\u0026iacute;, Heredia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cem\u003eTrypanosoma\u003c/em\u003e spp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e20.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003eAdult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003eInactive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003ch3\u003e\u003cbr\u003e\u003c/h3\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study provides the first molecular evidence of \u003cem\u003eLeishmania\u003c/em\u003e spp. in bats from Costa Rica, including the identification of \u003cem\u003eL. panamensis\u003c/em\u003e, and confirms the presence of \u003cem\u003eT. cruzi\u003c/em\u003e and \u003cem\u003eT. minasense\u003c/em\u003e through 18S rDNA analysis, highlighting the potential role of generalist bats in the eco-epidemiology of trypanosomatid parasites in the Neotropics. The \u003cem\u003eLeishmania\u003c/em\u003e infection rate found in bats in this study is comparable to the 3.7% reported in Brazil (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e) but lower than 9.1% observed in Venezuela (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e) and 8.9% reported in Mexico (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAmong the bat species found to harbor \u003cem\u003eLeishmania\u003c/em\u003e spp. in this study, \u003cem\u003eA. lituratus\u003c/em\u003e and \u003cem\u003eS. parvidens\u003c/em\u003e have previously been associated with \u003cem\u003eL. amazonensis\u003c/em\u003e in Brazil (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), while \u003cem\u003eA. jamaicensis\u003c/em\u003e, \u003cem\u003eA. lituratus\u003c/em\u003e, and \u003cem\u003eS. parvidens\u003c/em\u003e were reported with \u003cem\u003eL. mexicana\u003c/em\u003e in Mexico (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). The detection of \u003cem\u003eLeishmania\u003c/em\u003e spp. DNA in \u003cem\u003eC. castanea\u003c/em\u003e represents the first report of infection in this bat species.\u003c/p\u003e\u003cp\u003eThe geographic distribution of \u003cem\u003eLeishmania\u003c/em\u003e-positive bats spanned four different localities in three provinces (Lim\u0026oacute;n, Cartago, and Guanacaste). Positive bats were detected in endemic regions for \u003cem\u003eLeishmania\u003c/em\u003e spp. (e.g., Turrialba-Cartago and Talamanca-Lim\u0026oacute;n), where phlebotomine vectors have been reported (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e), and in non-endemic areas (e.g., Orosi-Cartago and Barra Honda-Guanacaste), where bats were captured in caves\u0026mdash;natural habitats for phlebotomine. These caves share climatic and ecological conditions with historically endemic regions, suggesting that bats in these areas may coexist with potential vectors of \u003cem\u003eLeishmania\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eA bat of the species \u003cem\u003eS. parvidens\u003c/em\u003e from Orosi was positive for \u003cem\u003eL. panamensis\u003c/em\u003e. This parasite is the primary etiological agent of cutaneous leishmaniasis in the Caribbean region of Costa Rica and is recognized as the leading cause of this disease in the country (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eNotably, most of the \u003cem\u003eLeishmania\u003c/em\u003e-positive bats were females. This may be explained by behavioral and ecological factors: males tend to fly higher and segregate, whereas females spend more time in roosts, caring for offspring, and flying at lower heights, increasing their likelihood of being captured with mist nets (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRegarding the infection rate of \u003cem\u003eTrypanosoma\u003c/em\u003e spp. detected in this study, this is comparable to the 10.2% prevalence reported in Panama (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), but lower than the 12.9% reported in Brazil (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), 36,5% in Ecuador (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e), and 61,2% in Colombia (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Conversely, it is higher than those 1,6% reported in Mexico (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e) and 2,6% in Texas, USA (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). These variations in prevalence could be attributed to differences in ecological conditions, host species, and whether sampling was conducted exclusively in endemic areas or across both endemic and non-endemic regions, as was the case in this study.\u003c/p\u003e\u003cp\u003eThis is the first study to report the presence of \u003cem\u003eT. minasense\u003c/em\u003e and \u003cem\u003eT. cruzi\u003c/em\u003e in bats from Costa Rica, and the first to confirm infection through molecular techniques (PCR and sequencing). Species found infected with \u003cem\u003eT. cruzi\u003c/em\u003e included \u003cem\u003eA. jamaicensis\u003c/em\u003e and \u003cem\u003eC. perspicillata\u003c/em\u003e, which have previously been reported as hosts in Panama (\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), as well as in Colombia, Brazil, and Ecuador (\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe detection of \u003cem\u003eT. minasense\u003c/em\u003e in \u003cem\u003eG. mutica\u003c/em\u003e and \u003cem\u003eC. sowelli\u003c/em\u003e represents the first report of this trypanosome in these bat species. \u003cem\u003eTrypanosoma minasense\u003c/em\u003e has previously been reported in other mammalian hosts, such as non-human primates, including \u003cem\u003eSaimiri sciureus\u003c/em\u003e imported to Japan from South America (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e) and in all four species of monkeys from Costa Rica, living in different provinces in captivity as in the wild (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Although its vector is still unknown, \u003cem\u003eT. minasense\u003c/em\u003e is currently considered non-pathogenic for bats (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe distribution of \u003cem\u003eTrypanosoma\u003c/em\u003e-positive bats extended across both Pacific and Caribbean coasts. Two \u003cem\u003eT. cruzi\u003c/em\u003e-positive individuals were captured in Puerto Viejo, Sarapiqu\u0026iacute;, an area where \u003cem\u003eT. dimidiata\u003c/em\u003e, the vector of Chagas disease, has been reported (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Another \u003cem\u003eT. cruzi\u003c/em\u003e-positive bat was found in Carara National Park, where \u003cem\u003eT. dimidiata\u003c/em\u003e has not yet been documented but where environmental conditions, such as altitude, temperature, and precipitation, are consistent with its known range (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Moreover, \u003cem\u003eT. dimidiata\u003c/em\u003e is known to inhabit ecotopes such as bat caves, rock piles, and tree hollows (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e), which coincide with the roosting habits of the bat species captured in this study (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). These findings warrant further investigation into the potential presence of \u003cem\u003eT. dimidiata\u003c/em\u003e in areas where \u003cem\u003eT. cruzi\u003c/em\u003e-positive bats were detected.\u003c/p\u003e\u003cp\u003e\u003cem\u003eT. minasense\u003c/em\u003e was exclusively detected on the Caribbean coast, in Orosi, Cartago province, and in K\u0026euml;koldi (Talamanca), and Corbana (Gu\u0026aacute;piles), Lim\u0026oacute;n province. Previous reports found \u003cem\u003eT. minasense\u003c/em\u003e in non-human primates from four provinces (Puntarenas, Alajuela, Lim\u0026oacute;n, and Heredia) in Costa Rica (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCapture sites for \u003cem\u003eTrypanosoma\u003c/em\u003e- and \u003cem\u003eLeishmania\u003c/em\u003e-positive bats were typically characterized by fragmented habitats, agricultural fields, disturbed forests, and urban areas like those reported by Cottontail et al. 2009 (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) and V\u0026iacute;quez-Rodr\u0026iacute;guez in 2015 (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e), who observed higher trypanosomatid infection rates in altered landscapes. Such environments are associated with high densities of generalist bat species and reduced overall bat diversity (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e), which could promote immunosuppression and increase contact with vectors, thereby enhancing parasite transmission (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). The impact of these hemoflagellates on the health of bats should be investigated, as well as their role in the maintenance of the parasite's life cycle.\u003c/p\u003e\u003cp\u003eHabitat loss and extreme bioclimate events are causing changes in the reproductive vector cycles and their geographic distribution (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). Considering growing anthropogenic pressure on tropical ecosystems and the complex ecological interactions involved in \u003cem\u003eLeishmania\u003c/em\u003e and \u003cem\u003eTrypanosoma\u003c/em\u003e transmission cycles, it is crucial to conduct integrative studies on hosts, vectors, and environmental variables. Such approaches will improve our understanding of how ecosystem changes influence hemoflagellate circulation in bats and their potential implications for public health and disease emergence.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study provides the first molecular evidence of \u003cem\u003eLeishmania\u003c/em\u003e spp., including \u003cem\u003eL. panamensis\u003c/em\u003e, as well as \u003cem\u003eT. cruzi\u003c/em\u003e and \u003cem\u003eT. minasense\u003c/em\u003e infections in bats from Costa Rica, underscoring the importance of these mammals as potential reservoirs or sentinels in the eco-epidemiology of trypanosomatid parasites. The detection of these parasites in both endemic and non-endemic areas, particularly within altered habitats, highlights the role of environmental and ecological factors\u0026mdash;such as habitat fragmentation, bat behavior, and possible vector presence\u0026mdash;in shaping transmission dynamics. These findings contribute to a better understanding of the distribution of trypanosomatid infections in the Neotropics and emphasize the need for integrated surveillance approaches that include the role of these mammals in disease transmission cycles and the impact of these parasites on the health of bats.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics and Animal Welfare\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures for the capture and sampling of bats in this study were approved by the Bioethics Committee of the School of Veterinary Medicine, Universidad Nacional, Costa Rica. The study was conducted under the corresponding permits granted by CONAGEBIO (National Commission for Biodiversity Management, File No. 163) and SINAC (National System of Conservation Areas).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analysed during the current study are available from the corresponding author on reasonable request.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Fondo Institucional de Desarrollo Acad\u0026eacute;mico (FIDA), Universidad Nacional, Project 0021-12: Murci\u0026eacute;lagos como bioindicadores: Presencia de agentes infecciosos \u0026nbsp;(virus, hongos y par\u0026aacute;sitos) en murci\u0026eacute;lagos en Costa Rica\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by RR, AUV, KSM, MJZM, and GD. The first draft of the manuscript was written by RR, AUV, and GD, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMedell\u0026iacute;n RA, Gaona O. Seed dispersal by bats and birds in forest and disturbed habitats in Chiapas, M\u0026eacute;xico. Biotropica. 1999;31:432-41.\u003c/li\u003e\n\u003cli\u003eMeyer CF, Fr\u0026uuml;nd J, Lizano WP, Kalko EK. Ecological correlates of vulnerability to fragmentation in Neotropical bats. J Appl Ecol. 2008;45(1):381-91.\u003c/li\u003e\n\u003cli\u003eSerra-Cobo J, Lopez-Roig M. Bats and emerging infections: an ecological and virological puzzle. In: Emerging and Re-emerging Viral Infections. Cham: Springer; 2016. p. 35\u0026ndash;48.\u003c/li\u003e\n\u003cli\u003eRangel DA, Lisboa CV, Novaes RLM, Silva BA, Souza RDF, Jansen AM, et al. Isolation and characterization of trypanosomatids, including Crithidia mellificae, in bats from the Atlantic Forest of Rio de Janeiro, Brazil. PLoS Negl Trop Dis. 2019;13(7):e0007527.\u003c/li\u003e\n\u003cli\u003eRotureau B. Ecology of the \u003cem\u003eLeishmania\u003c/em\u003e species in the Guianan ecoregion complex. Am J Trop Med Hyg. 2006;74(1):81\u0026ndash;6. https://doi.org/10.4269/ajtmh.2006.74.81\u003c/li\u003e\n\u003cli\u003eJansen AM, Roque ALR. Domestic and Wild Mammalian Reservoirs. In: American Trypanosomiasis. 2010;249\u0026ndash;76. https://doi.org/10.1016/B978-0-12-384876-5.00011-333\u003c/li\u003e\n\u003cli\u003eShipley R, Wright E, Selden D, Wu G, Aegerter J, Fooks AR, et al. Bats and viruses: emergence of novel lyssaviruses and association of bats with viral zoonoses in the EU. Trop Med Infect Dis. 2019;4(1):31.\u003c/li\u003e\n\u003cli\u003eSchneider M, Burgoa C. Algunas consideraciones sobre la rabia humana transmitida por murci\u0026eacute;lago. Salud Publica Mex. 1995;37(4):354\u0026ndash;62.\u003c/li\u003e\n\u003cli\u003eWang LF, Shi Z, Zhang S, Field H, Daszak P, Eaton BT. Review of bats and SARS. Emerg Infect Dis. 2006;12:1834\u0026ndash;40.\u003c/li\u003e\n\u003cli\u003eCalisher CH, Holmes KV, Dominguez SR, Schountz T, Cryan P. Bats prove to be rich reservoirs for emerging viruses. Microbe. 2008;3:521-8.\u003c/li\u003e\n\u003cli\u003eHui-Ju H, Hong-Ling W, Chuan-Min Z, Fang-Fang C, Li-Mei L, Jian-Wei L, et al. Los murci\u0026eacute;lagos como reservorios de enfermedades infecciosas emergentes graves. Virus Res. 2015;205:1-6. https://doi.org/10.1016/j.virusres.2015.05.006\u003c/li\u003e\n\u003cli\u003eM\u0026uuml;hldorfer K. Bats and bacterial pathogens: a review. Zoonoses Public Health. 2013;60(1):93-103.\u003c/li\u003e\n\u003cli\u003eTorres-Castro M, Febles-Sol\u0026iacute;s V, Hern\u0026aacute;ndez-Betancourt S, Noh-Pech H, Estrella E, Pel\u0026aacute;ez-S\u0026aacute;nchez R, et al. Leptospira pat\u0026oacute;genas en murci\u0026eacute;lagos de Campeche y Yucat\u0026aacute;n, M\u0026eacute;xico. Rev MVZ C\u0026oacute;rdoba. 2020;25:e1815. https://doi.org/10.21897/rmvz.1815\u003c/li\u003e\n\u003cli\u003ePanizo MM, Dolande M, Reviakina V, Maldonado B. Histoplasmosis pulmonar asociada con visita a cuevas: Descripci\u0026oacute;n de un brote epid\u0026eacute;mico y revisi\u0026oacute;n de la literatura. Rev Soc Venez Microbiol. 2001;21(1):30-5. \u003c/li\u003e\n\u003cli\u003eHodo CL, Goodwin CC, Mayes BC, Mariscal JA, Waldrup KA, Hamer SA. Trypanosome species, including \u003cem\u003eTrypanosoma\u003c/em\u003e \u003cem\u003ecruzi\u003c/em\u003e, in sylvatic and peridomestic bats of Texas, USA. Acta Trop. 2016;164:259-66.\u003c/li\u003e\n\u003cli\u003eJansen AM, das Chagas Xavier SC, Roque ALR. \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e transmission in the wild and its most important reservoir hosts in Brazil. Parasites Vectors. 2018;11(1):1-25.\u003c/li\u003e\n\u003cli\u003eKassahun A, Sadlova J, Benda P, Kostalova T, Warburg A, Hailu A, et al. Natural infection of bats with \u003cem\u003eLeishmania \u003c/em\u003ein Ethiopia. Acta Trop. 2015. http://dx.doi.org/10.1016/j.actatropica.2015.07.024\u003c/li\u003e\n\u003cli\u003eLima HD, Rodr\u0026iacute;guez N, Barrios M, \u0026Aacute;vila \u0026Aacute;, Ca\u0026ntilde;izales I, Guti\u0026eacute;rrez S. Isolation and molecular identification of \u003cem\u003eLeishmania chagasi\u003c/em\u003e from a bat (Carollia perspicillata) in northeastern Venezuela. Mem Inst Oswaldo Cruz. 2008;103:412-4.\u003c/li\u003e\n\u003cli\u003eSavani ES, de Almeida MF, De Oliveira Camargo MC, D\u0026rsquo;Auria SR, Silva MM, de Oliveira ML, et al. Detection of \u003cem\u003eLeishmania amazonensis\u003c/em\u003e and \u003cem\u003eLeishmania infantum chagasi\u003c/em\u003e in Brazilian bats. Vet Parasitol. 2010;168:5\u0026ndash;10.\u003c/li\u003e\n\u003cli\u003eShapiro JT, da Costa Lima Junior MS, Dorval MEC, de Oliveira Fran\u0026ccedil;a A, Cepa Matos MDF, Bordignon MO. First record of\u003cem\u003e Leishmania braziliensis\u003c/em\u003e presence detected in bats, Mato Grosso do Sul, southwest Brazil. Acta Trop. 2013;128:171\u0026ndash;4.\u003c/li\u003e\n\u003cli\u003eCastro LS, Dorval ME, Matheus LM, Bednaski AV, Facco GG, Silveira M, et al. \u003cem\u003eLeishmania\u003c/em\u003e presence in bats in areas endemic for leishmaniasis in central-west Brazil. Int J Parasitol Parasites Wildl. 2020;11:261-7.\u003c/li\u003e\n\u003cli\u003eHoare CA. The Trypanosomes of Mammals. A Zoological Monograph. Oxford and Edinburgh: Blackwell Scientific Publications; 1972.\u003c/li\u003e\n\u003cli\u003eCottontail VM, Wellinghausen N, Kalko EKV. Habitat fragmentation and haemoparasites in the common fruit bats Artibeus jamaicensis (Phyllostomidae) in tropical lowland forest in Panam\u0026aacute;. Parasitology. 2009;136(10):1133-45.\u003c/li\u003e\n\u003cli\u003eCottontail VM, Kalko EK, Cottontail I, Wellinghausen N, Tschapka M, Perkins SL, et al. High local diversity of \u003cem\u003eTrypanosoma\u003c/em\u003e in a common bat species, and implications for the biogeography and taxonomy of the T. cruzi clade. PLoS One. 2014;9:e108603.\u003c/li\u003e\n\u003cli\u003eTorres-Castro M, Cuevas-Koh N, Hern\u0026aacute;ndez-Betancourt S, Noh-Pech H, Estrella E, Herrera-Flores B, et al. Natural infection with \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e in bats captured in Campeche and Yucat\u0026aacute;n, M\u0026eacute;xico. Biom\u0026eacute;dica. 2021;41(Supl.1):131\u0026ndash;40. https://doi.org/10.7705/biomedica.5450\u003c/li\u003e\n\u003cli\u003eMoo-Millan JI, Tu W, Montalvo-Balam TJ, Ibarra-L\u0026oacute;pez MP, Hern\u0026aacute;ndez-Betancourt S, May-Concha IJ, et al. Presence of \u003cem\u003eTrypanosoma cruzi \u003c/em\u003eTcI and \u003cem\u003eTrypanosoma dionisii\u003c/em\u003e in sylvatic bats from Yucatan, Mexico. Trans R Soc Trop Med Hyg. 2024;trae023.\u003c/li\u003e\n\u003cli\u003eFran\u0026ccedil;a DA, Louro M, Z\u0026uacute;quete S, Zanini DS, Moraes GN, Rocha GS, et al. \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e in Bats (Chiroptera; Mammalia) from the Brazilian Atlantic Forest, S\u0026atilde;o Paulo State. Microorganisms. 2024;12:945. https://doi.org/10.3390/microorganisms12050945\u003c/li\u003e\n\u003cli\u003eBrun R, Hecker H, Lun ZR. \u003cem\u003eTrypanosoma evansi\u003c/em\u003e and \u003cem\u003eT. equiperdum\u003c/em\u003e: distribution, biology, treatment and phylogenetic relationship (a review). Vet Parasitol. 1998;79(2):95-107.\u003c/li\u003e\n\u003cli\u003eSilva-Iturriza A, Nassar JM, Garcia-Rawlins AM, Rosales R, Mijares A. \u003cem\u003eTrypanosoma evansi \u003c/em\u003ekDNA minicircle found in the Venezuelan nectar-feeding bat Leptonycteris curasoae (Glossophaginae), supports the hypothesis of multiple origins of that parasite in South America. Parasitol Int. 2013;62(2):95\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eTorrico MC, T\u0026eacute;llez T, Tenorio O, Rojas L, Huaranca JC, De la Barra A, et al. Tripanosom\u0026aacute;tidos aislados de mam\u0026iacute;feros silvestres en tres departamentos de Bolivia (Cochabamba, Potos\u0026iacute; y Santa Cruz de la Sierra). Gac Med Boliv. 2013;36(1):6\u0026ndash;10.\u003c/li\u003e\n\u003cli\u003eZeled\u0026oacute;n R, Vieto PL. Hallazgo de Schizotrypanum vespertilionis (Battaglia, 1904) en la sangre de murci\u0026eacute;lagos de Costa Rica. Rev Biol Trop. 1957;5:123\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eZeled\u0026oacute;n R, Rosabal R. \u003cem\u003eTrypanosoma leonidasdeanei\u003c/em\u003e sp. nov. in insectivorous bats of Costa Rica. Ann Trop Med Parasitol. 1969;63:221\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eJaramillo-Antill\u0026oacute;n O, Espinoza-Aguirre A, Lobo-Philp R. Estado actual de la leishmaniosis en Costa Rica. Acta Med Costarric. 2009;51:158\u0026ndash;64.\u003c/li\u003e\n\u003cli\u003eZeled\u0026oacute;n R, et al. Chagas disease in Costa Rica. Am J Trop Med Hyg. 1975;24(3):405\u0026ndash;408.\u003c/li\u003e\n\u003cli\u003eM\u0026uuml;ller E. Investigaciones en frutos y semillas de \u0026aacute;rboles individuales de cinco especies forestales de la Regi\u0026oacute;n Huetar Norte de Costa Rica, con especial consideraci\u0026oacute;n en el almacenamiento. Documento 51. COSEFORMA, Costa Rica; 1997. 237 p.\u003c/li\u003e\n\u003cli\u003eTimm RM, Laval RK, Rodr\u0026iacute;guez B. Clave de Campo para los Murci\u0026eacute;lagos de Costa Rica. Brenesia. 1999;52:1\u0026ndash;32.\u003c/li\u003e\n\u003cli\u003eLaval RK, Rodr\u0026iacute;guez-Herrera B. Murci\u0026eacute;lagos de Costa Rica. Santo Domingo, Heredia, Costa Rica: Editorial INBio; 2002. 310 p.\u003c/li\u003e\n\u003cli\u003eSchinnerl M, Aydinonat D, Schwarzenberger F, Voigt C. Hematological survey of common neotropical bat species from Costa Rica. J Zoo Wildl Med. 2011;42:382\u0026ndash;91.\u003c/li\u003e\n\u003cli\u003eMedeiros ACR, Rodr\u0026iacute;guez SS, Roselino AMF. Comparison of the specificity of PCR and the histopathological detection of \u003cem\u003eLeishmania\u003c/em\u003e for the diagnosis of American cutaneous leishmaniasis. Braz J Med Biol Res. 2002;35(4):421-4.\u003c/li\u003e\n\u003cli\u003eEl Tai NO, El Fari M, Mauricio I, Miles MA, Oskam L, El Safi SH, Presber WH, Sch\u0026ouml;nian G. \u003cem\u003eLeishmania donovani\u003c/em\u003e: intraspecific polymorphisms of Sudanese isolates revealed by PCR-based analyses and DNA sequencing. Exp Parasitol. 2001 Jan;97(1):35-44. doi:10.1006/expr.2001.4592. PMID: 11207112.\u003c/li\u003e\n\u003cli\u003eV\u0026iacute;quez-Rodr\u0026iacute;guez. Prevalencia de \u003cem\u003eLeishmania mexicana \u003c/em\u003ey \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e en los murci\u0026eacute;lagos \u003cem\u003eCarollia sowelli\u003c/em\u003e y\u003cem\u003e Sturnira lilium \u003c/em\u003ebajo dos condiciones distintas de perturbaci\u0026oacute;n antropog\u0026eacute;nica en la Selva Lacandona, Chiapas. [Tesis de Maestr\u0026iacute;a]. M\u0026eacute;xico: Universidad Nacional Aut\u0026oacute;noma de M\u0026eacute;xico; 2015.\u003c/li\u003e\n\u003cli\u003eBerzunza-Cruz M, Rodr\u0026iacute;guez-Moreno \u0026Aacute;, Guti\u0026eacute;rrez-Granados G, Gonz\u0026aacute;lez-Salazar C, Stephens CR, Hidalgo-Mihart M, et al. \u003cem\u003eLeishmania (L.) mexicana \u003c/em\u003einfected bats in Mexico: novel potential reservoirs. PLoS Negl Trop Dis. 2015;9(1):e0003438.\u003c/li\u003e\n\u003cli\u003eZeled\u0026oacute;n R, Murillo J, Guti\u0026eacute;rrez H. Fleb\u0026oacute;tomos antrop\u0026oacute;filos y leishmaniasis cut\u0026aacute;nea en Costa Rica. Bol Oficina Sanit Panam. 1985;99:163\u0026ndash;72.\u003c/li\u003e\n\u003cli\u003ePeraza J, Urbina A, Zeled\u0026oacute;n R. Zymodeme and serodeme characterization of \u003cem\u003eLeishmania\u003c/em\u003e isolates obtained from Costa Rican patients. Mem Inst Oswaldo Cruz. 1998;93(3):283-7.\u003c/li\u003e\n\u003cli\u003eCavazzana M, Marcili A, Lima L, da Silva FM, Junqueira \u0026Acirc;C, Veludo HH, et al. Phylogeographical, ecological and biological patterns shown by nuclear (ssrRNA and gGAPDH) and mitochondrial (Cyt b) genes of trypanosomes of the subgenus Schizotrypanum parasitic in Brazilian bats. Int J Parasitol. 2010;40(3):345-55.\u003c/li\u003e\n\u003cli\u003ePinto CM, Oca\u0026ntilde;a-Mayorga S, Tapia EE, Lobos SE, Zurita AP, Aguirre-Villac\u0026iacute;s F, et al. Bats, trypanosomes, and triatomines in Ecuador: new insights into the diversity, transmission, and origins of \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e and Chagas disease. PLoS One. 2015;10:e0139999. http://dx.doi.org/10.1371/journal.pone.0139999\u003c/li\u003e\n\u003cli\u003eRam\u0026iacute;rez JD, Tapia-Calle G, Mu\u0026ntilde;oz-Cruz G, Poveda C, Rend\u0026oacute;n LM, Hincapi\u0026eacute; E, Guhl F. \u003cem\u003eTrypanosoma \u003c/em\u003especies in neo-tropical bats: biological, evolutionary and epidemiological implications. Infect Genet Evol. 2014;22:250-6.\u003c/li\u003e\n\u003cli\u003eSato TM, Passos FC, Nogueira AC. Frugivoria de morcegos (Mammalia, Chiroptera) em \u003cem\u003eCecropia pachystachya\u003c/em\u003e (Urticaceae) e seus efeitos na germina\u0026ccedil;\u0026atilde;o de sementes. \u003cem\u003ePap Avulsos Zool.\u003c/em\u003e 2008;48(3):19\u0026ndash;26.\u003c/li\u003e\n\u003cli\u003eChinchilla M, Troyo A, Guerrero OM, Guti\u0026eacute;rrez-Espeleta GA, S\u0026aacute;nchez R. Presencia de \u003cem\u003eTrypanosoma minasense\u003c/em\u003e (Kinetoplastida: Trypanosomatidae) en Alouatta palliata (Primates: Cebidae) de Costa Rica. Parasitol Latinoam. 2005;60(1-2):90-2.\u003c/li\u003e\n\u003cli\u003eCottontail VM, Kalko EKV, Cottontail I, Wellinghausen N, Tschapka M, et al. High local diversity of \u003cem\u003eTrypanosoma\u003c/em\u003e in a common bat species, and implications for the biogeography and taxonomy of the \u003cem\u003eT. cruzi\u003c/em\u003e clade. PLoS One. 2014;9(9):e108603. doi:10.1371/journal.pone.0108603. \u003c/li\u003e\n\u003cli\u003eZeled\u0026oacute;n R, Calvo N, Montenegro VM, Lorosa ES, Ar\u0026eacute;valo C. A survey on \u003cem\u003eTriatoma dimidiata \u003c/em\u003ein an urban area of the province of Heredia, Costa Rica. Mem Inst Oswaldo Cruz. 2005;100(6):507-12. \u003c/li\u003e\n\u003cli\u003eZeled\u0026oacute;n R. \u003cem\u003eTriatoma dimidiata \u003c/em\u003ey su relaci\u0026oacute;n con la enfermedad de Chagas\u003cem\u003e.\u003c/em\u003e San Jos\u0026eacute; (CR): EUNED; 1981. 164 p.\u003c/li\u003e\n\u003cli\u003eSolorzano-Morales A, Tien C, Alfaro S, Dolz G. Trypanosomatid infections in non-human primates of Costa Rica. In: IV Bienal WDA Latinoam\u0026eacute;rica; 2019 Jul 16\u0026ndash;18; San Jos\u0026eacute;, Costa Rica.\u003c/li\u003e\n\u003cli\u003eFenton MB, Acharya L, Audet D, Hickey MBC, Merriman C, Obrist MK, Adkins B. Phyllostomid bats (Chiroptera: Phyllostomidae) as indicators of habitat disruption in the Neotropics. Biotropica. 1992;24(3):440-6\u003cstrong\u003e.\u003c/strong\u003e\u003c/li\u003e\n\u003cli\u003eFran\u0026ccedil;a DA, Louro M, Z\u0026uacute;quete S, Zanini DS, Moraes GN, Rocha GS, Biondo LM, Fornazari F, Menozzi BD, Fonseca IP, et al. \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e in bats (Chiroptera; Mammalia) from the Brazilian Atlantic Forest, S\u0026atilde;o Paulo State. Microorganisms. 2024;12(5):945. doi:10.3390/microorganisms12050945.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Leishmania spp., Trypanosoma spp., wildlife, polymerase chain reaction","lastPublishedDoi":"10.21203/rs.3.rs-7466101/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7466101/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Hemoflagellates of the genera \u003cem\u003eLeishmania\u003c/em\u003e and \u003cem\u003eTrypanosoma\u003c/em\u003e have been reported in bats from various geographic regions worldwide. In Costa Rica, the presence of trypanosomatids in bats has been limited to descriptions of \u003cem\u003eTrypanosoma vespertilionis\u003c/em\u003e and \u003cem\u003eTrypanosoma leonidasdeanei\u003c/em\u003e in the 1950s and 1960s, with no prior reports of \u003cem\u003eLeishmania\u003c/em\u003e spp. in these mammals. This study aimed to detect trypanosomatid hemoflagellates in generalist bat species from Costa Rica.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Between June 2013 and August 2014, a total of 194 bats were captured from 11 localities across five provinces. Blood samples were collected from 98 individuals and analyzed by polymerase chain reaction (PCR) for the presence of \u003cem\u003eLeishmania\u003c/em\u003e spp. and \u003cem\u003eTrypanosoma\u003c/em\u003e spp.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Four samples (4.1%) tested positive for \u003cem\u003eLeishmania\u003c/em\u003espp. and nine samples (9.2%) for \u003cem\u003eTrypanosoma\u003c/em\u003espp. Sequencing determined for the first time the presence of \u003cem\u003eLeishmania panamensis\u003c/em\u003e and \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e in generalist bats from Costa Rica, and confirmed the presence of \u003cem\u003eTrypanosoma minasense\u003c/em\u003e in these volant mammals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e These findings underscore the importance of integrative studies involving hosts, vectors, and environmental factors to understand the impact of ecosystem changes on trypanosomatid distribution and their potential implications for public health. It is necessary to understand the role that these hemoflagellates play on the health of bats, the importance of these mammals in leishmaniasis and trypanosomiasis, the transmission cycles and the ecological and bioclimate factors associated with vector and wildlife dynamics.\u003c/p\u003e","manuscriptTitle":"Detection of Trypanosomatid hemoflagellates in generalist bats from Costa Rica","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-10 08:56:11","doi":"10.21203/rs.3.rs-7466101/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aa62c0ae-1e29-4da9-96fd-14c645d164a5","owner":[],"postedDate":"September 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-21T16:53:19+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-10 08:56:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7466101","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7466101","identity":"rs-7466101","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}