Endosimbiontes bacterianos y microbiota intestinal de vectores de Leishmania y otros tripanosomátidos del departamento de Sucre, región Caribe de Colombia
Introducción y justificación. Los flebotominos son dípteros de gran relevancia en salud pública por su papel como vectores transmisores de diversos patógenos como Leishmania, Bartonella, Vesiculovirus, y Phlebovirus. Pese a la importancia epidemiológica de los flebotominos, no existen métodos de con...
- Autores:
-
Alemán Santos, Maira Alejandra
- Tipo de recurso:
- Fecha de publicación:
- 2025
- Institución:
- Universidad de Córdoba
- Repositorio:
- Repositorio Institucional Unicórdoba
- Idioma:
- spa
- OAI Identifier:
- oai:repositorio.unicordoba.edu.co:ucordoba/9134
- Acceso en línea:
- https://repositorio.unicordoba.edu.co/handle/ucordoba/9134
https://repositorio.unicordoba.edu.co/
- Palabra clave:
- Endosimbiontes
Vector
Flebotominos
Agentes de control biológico
Leishmania
Trypanosomatidae
Secuenciación MinION
Endosymbionts
Vector
Phlebotomine sandflies
Biological control agents
Leishmania
Trypanosomatidae
MinION sequencing
- Rights
- embargoedAccess
- License
- Copyright Universidad de Córdoba, 2025
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Endosimbiontes bacterianos y microbiota intestinal de vectores de Leishmania y otros tripanosomátidos del departamento de Sucre, región Caribe de Colombia |
| title |
Endosimbiontes bacterianos y microbiota intestinal de vectores de Leishmania y otros tripanosomátidos del departamento de Sucre, región Caribe de Colombia |
| spellingShingle |
Endosimbiontes bacterianos y microbiota intestinal de vectores de Leishmania y otros tripanosomátidos del departamento de Sucre, región Caribe de Colombia Endosimbiontes Vector Flebotominos Agentes de control biológico Leishmania Trypanosomatidae Secuenciación MinION Endosymbionts Vector Phlebotomine sandflies Biological control agents Leishmania Trypanosomatidae MinION sequencing |
| title_short |
Endosimbiontes bacterianos y microbiota intestinal de vectores de Leishmania y otros tripanosomátidos del departamento de Sucre, región Caribe de Colombia |
| title_full |
Endosimbiontes bacterianos y microbiota intestinal de vectores de Leishmania y otros tripanosomátidos del departamento de Sucre, región Caribe de Colombia |
| title_fullStr |
Endosimbiontes bacterianos y microbiota intestinal de vectores de Leishmania y otros tripanosomátidos del departamento de Sucre, región Caribe de Colombia |
| title_full_unstemmed |
Endosimbiontes bacterianos y microbiota intestinal de vectores de Leishmania y otros tripanosomátidos del departamento de Sucre, región Caribe de Colombia |
| title_sort |
Endosimbiontes bacterianos y microbiota intestinal de vectores de Leishmania y otros tripanosomátidos del departamento de Sucre, región Caribe de Colombia |
| dc.creator.fl_str_mv |
Alemán Santos, Maira Alejandra |
| dc.contributor.advisor.none.fl_str_mv |
Paternina Tuirán, Luis Enrique Mattar Velilla, Salim |
| dc.contributor.author.none.fl_str_mv |
Alemán Santos, Maira Alejandra |
| dc.contributor.jury.none.fl_str_mv |
Lozano Sardaneta, Yokomi Nisei Maestre Serrano, Ronald Hoyos López, Richard |
| dc.subject.proposal.spa.fl_str_mv |
Endosimbiontes Vector Flebotominos Agentes de control biológico Leishmania Trypanosomatidae Secuenciación MinION |
| topic |
Endosimbiontes Vector Flebotominos Agentes de control biológico Leishmania Trypanosomatidae Secuenciación MinION Endosymbionts Vector Phlebotomine sandflies Biological control agents Leishmania Trypanosomatidae MinION sequencing |
| dc.subject.keywords.eng.fl_str_mv |
Endosymbionts Vector Phlebotomine sandflies Biological control agents Leishmania Trypanosomatidae MinION sequencing |
| description |
Introducción y justificación. Los flebotominos son dípteros de gran relevancia en salud pública por su papel como vectores transmisores de diversos patógenos como Leishmania, Bartonella, Vesiculovirus, y Phlebovirus. Pese a la importancia epidemiológica de los flebotominos, no existen métodos de control efectivo debido a sus peculiares características. Los esfuerzos para el control de la leishmaniasis se han centrado en el diagnóstico y el tratamiento de la enfermedad. Esto ha motivado la implementación de medidas alternativas hacia el flebotomino para el control de la enfermedad, basándose en las relaciones antagónicas de algunas bacterias del tracto digestivo del vector y los parásitos del género Leishmania. No obstante, su aplicación para el control de la leishmaniasis es limitada por la falta de conocimiento sobre la frecuencia con la que se presentan estas interacciones en la naturaleza. El control biológico con la bacteria Wolbachia ha sido usada exitosamente para mitigar enfermedades virales en los mosquitos portadores de los arbovirus Dengue y Chikungunya. También, este endosimbionte interfiere con el establecimiento del apicomplexo Plasmodium. En este escenario, las herramientas de metagenómica ofrecen la oportunidad de caracterizar el bacterioma de los vectores, para establecer las posibles relaciones antagónicas que protejan a los insectos de la infección con los parásitos. Objetivo. Caracterizar los endosimbiontes bacterianos y la microbiota intestinal en vectores de Leishmania y otros tripanosomátidos en el departamento de Sucre, región Caribe de Colombia. Métodos. Estudio de tipo descriptivo prospectivo realizado entre noviembre 2021 y febrero 2023 en el departamento de Sucre en los municipios de Sincelejo, Colosó, Sampués y Ovejas. Mediante búsqueda activa, trampas Shannon y CDC-light se realizaron muestreos para la recolección de flebotominos. Las hembras recolectadas se mantuvieron vivas hasta su disección para la identificación taxonómica y la búsqueda microscópica de flagelados en el tracto intestinal. |
| publishDate |
2025 |
| dc.date.accessioned.none.fl_str_mv |
2025-04-24T18:11:57Z |
| dc.date.available.none.fl_str_mv |
2025-04-24T18:11:57Z 2026-04-24 |
| dc.date.issued.none.fl_str_mv |
2025-04-24 |
| dc.type.none.fl_str_mv |
Trabajo de grado - Doctorado |
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info:eu-repo/semantics/masterThesis |
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info:eu-repo/semantics/acceptedVersion |
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Text |
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http://purl.org/redcol/resource_type/TM |
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acceptedVersion |
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https://repositorio.unicordoba.edu.co/handle/ucordoba/9134 |
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Universidad de Córdoba |
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Repositorio Universidad de Córdoba |
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https://repositorio.unicordoba.edu.co/ |
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https://repositorio.unicordoba.edu.co/handle/ucordoba/9134 https://repositorio.unicordoba.edu.co/ |
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Universidad de Córdoba Repositorio Universidad de Córdoba |
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spa |
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Using Proteomics to Understand How Leishmania Parasites Survive inside the Host and Establish Infection. International Journal of Molecular Sciences, 17(8), 1270. https://doi.org/10.3390/ijms17081270 Villarreal Julio, R. G., Herrera, G., Muskus López, C. E., Villarreal Julio, R. G., Herrera, G., & Muskus López, C. E. (2021). Identificación de especies de Leishmania mediante PCR en tiempo real acoplada a curvas de fusión de alta resolución. Revista Cubana de Medicina Tropical, 73(3). http://scielo.sld.cu/scielo.php?script=sci_abstract&pid=S0375-076020210003 00007&lng=es&nrm=iso&tlng=es Vivero-Gomez, R. J., Cadavid-Restrepo, G., Herrera, C. X. M., & Soto, S. I. U. (2017). Molecular detection and identification of Wolbachia in three species of the genus Lutzomyia on the Colombian Caribbean coast. Parasites & Vectors, 10(1), 110. https://doi.org/10.1186/s13071-017-2031-x Vivero-Gomez, R. J., Castañeda-Monsalve, V. A., Atencia, M. C., Hoyos-Lopez, R., Hurst, G. D., Cadavid-Restrepo, G., & Moreno-Herrera, C. X. (2021). Molecular phylogeny of heritable symbionts and microbiota diversity analysis in phlebotominae sand flies and Culex nigripalpus from Colombia. PLOS Neglected Tropical Diseases, 15(12), e0009942. https://doi.org/10.1371/journal.pntd.0009942 Vivero-Gomez, R. J., Castañeda-Monsalve, V. A., Romero, L. R., D. Hurst, G., Cadavid-Restrepo, G., & Moreno-Herrera, C. X. (2021). Gut Microbiota Dynamics in Natural Populations of Pintomyia evansi under Experimental Infection with Leishmania infantum. Microorganisms, 9(6), 1214. https://doi.org/10.3390/microorganisms9061214 Vivero-Gomez, R. J., Gutiérrez-García, M., Moreno-Herrera, C. X., Cadavid-Restrepo, G., & Uribe-Soto, S. (2016). Presencia de Wolbachia y Leishmania en una población de Lutzomyia evansi de la Costa Caribe de Colombia. Revista de la Facultad de Ciencias, 5(2), Article 2. https://doi.org/10.15446/rev.fac.cienc.v5n2.56684 Vivero-Gomez, R. J., Jaramillo, N. G., Cadavid-Restrepo, G., Soto, S. I. U., & Herrera, C. X. M. (2016). Structural differences in gut bacteria communities in developmental stages of natural populations of Lutzomyia evansi from Colombia’s Caribbean coast. Parasites & Vectors, 9(1), 496. https://doi.org/10.1186/s13071-016-1766-0 Vivero-Gomez, R. J., Villegas-Plazas, M., Cadavid-Restrepo, G. E., Herrera, C. X. M., Uribe, S. I., & Junca, H. (2019). Wild specimens of sand fly phlebotomine Lutzomyia evansi, vector of leishmaniasis, show high abundance of Methylobacterium and natural carriage of Wolbachia and Cardinium types in the midgut microbiome. Scientific Reports, 9(1), 17746. https://doi.org/10.1038/s41598-019-53769-z Votýpka, J., Zeman, Š., Stříbrná, E., Pajer, P., Bartoš, O., Kment, P., Iv, J. L., & Iii, J. L. (2024). Multiple and frequent trypanosomatid co-infections of insects: The Cuban case study. Parasitology, 151(6), 567-578. https://doi.org/10.1017/S0031182024000453 Wallace, F. G. (1966). 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Isolations of Leishmania braziliensis (Kinetoplastida: Trypanosomatidae) from Cryopreserved Colombian Sand Flies (Diptera: Psychodidae). Journal of Medical Entomology, 24(5), 587-589. https://doi.org/10.1093/jmedent/24.5.587 Yun, J.-H., Roh, S. W., Whon, T. W., Jung, M.-J., Kim, M.-S., Park, D.-S., Yoon, C., Nam, Y.-D., Kim, Y.-J., Choi, J.-H., Kim, J.-Y., Shin, N.-R., Kim, S.-H., Lee, W.-J., & Bae, J.-W. (2014). Insect Gut Bacterial Diversity Determined by Environmental Habitat, Diet, Developmental Stage, and Phylogeny of Host. Applied and Environmental Microbiology, 80(17), 5254-5264. https://doi.org/10.1128/AEM.01226-14 Zambrano Hernández, C. P., Ayala Sotelo, M. S., Fuya Oviedo, O. P., Barraza, O. C., & Rodríguez Toro, G. (2016). Cartagena: Nuevo foco de leishmaniasis visceral urbana en Colombia. Ciencia en Desarrollo, 7(1), 83-91. https://doi.org/10.19053/01217488.4234 Zchori‐Fein, E., & Perlman, S. J. (2004). Distribution of the bacterial symbiont Cardinium in arthropods. Molecular Ecology, 13(7), 2009-2016. https://doi.org/10.1111/j.1365-294X.2004.02203.x Zhou, W., Rousset, F., & O’Neill, S. (1998). Phylogeny and PCR–based classification of Wolbachia strains using wsp gene sequences. Proceedings of the Royal Society of London. Series B: Biological Sciences, 265(1395), 509-515. https://doi.org/10.1098/rspb.1998.0324 |
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Paternina Tuirán, Luis Enrique2cad8246-576b-4a73-b024-3fce5a5eefa3-1Mattar Velilla, Salime905313b-2584-4269-bfdb-58d23418144a-1Alemán Santos, Maira Alejandra49e4a4f7-cd7d-44f3-a6ea-dd5d2231acee-1Lozano Sardaneta, Yokomi Nisei8eaeb7b5-50ce-4a8c-ab4b-75b2653bd3e8-1Maestre Serrano, Ronaldafea08a4-c18c-4734-a3d3-7aab6f4e80ff-1Hoyos López, Richardd12cc16d-9dd4-44ea-a059-d3659ffa1be4-12025-04-24T18:11:57Z2026-04-242025-04-24T18:11:57Z2025-04-24https://repositorio.unicordoba.edu.co/handle/ucordoba/9134Universidad de CórdobaRepositorio Universidad de Córdobahttps://repositorio.unicordoba.edu.co/Introducción y justificación. Los flebotominos son dípteros de gran relevancia en salud pública por su papel como vectores transmisores de diversos patógenos como Leishmania, Bartonella, Vesiculovirus, y Phlebovirus. Pese a la importancia epidemiológica de los flebotominos, no existen métodos de control efectivo debido a sus peculiares características. Los esfuerzos para el control de la leishmaniasis se han centrado en el diagnóstico y el tratamiento de la enfermedad. Esto ha motivado la implementación de medidas alternativas hacia el flebotomino para el control de la enfermedad, basándose en las relaciones antagónicas de algunas bacterias del tracto digestivo del vector y los parásitos del género Leishmania. No obstante, su aplicación para el control de la leishmaniasis es limitada por la falta de conocimiento sobre la frecuencia con la que se presentan estas interacciones en la naturaleza. El control biológico con la bacteria Wolbachia ha sido usada exitosamente para mitigar enfermedades virales en los mosquitos portadores de los arbovirus Dengue y Chikungunya. También, este endosimbionte interfiere con el establecimiento del apicomplexo Plasmodium. En este escenario, las herramientas de metagenómica ofrecen la oportunidad de caracterizar el bacterioma de los vectores, para establecer las posibles relaciones antagónicas que protejan a los insectos de la infección con los parásitos. Objetivo. Caracterizar los endosimbiontes bacterianos y la microbiota intestinal en vectores de Leishmania y otros tripanosomátidos en el departamento de Sucre, región Caribe de Colombia. Métodos. Estudio de tipo descriptivo prospectivo realizado entre noviembre 2021 y febrero 2023 en el departamento de Sucre en los municipios de Sincelejo, Colosó, Sampués y Ovejas. Mediante búsqueda activa, trampas Shannon y CDC-light se realizaron muestreos para la recolección de flebotominos. Las hembras recolectadas se mantuvieron vivas hasta su disección para la identificación taxonómica y la búsqueda microscópica de flagelados en el tracto intestinal. Introduction & Justification. Sand flies are highly relevant to public health because they are vectors of pathogens such as Leishmania, Bartonella, Vesiculovirus, and Phlebovirus. Despite the epidemiological importance of sandflies, there are no effective control methods for them owing to their unique characteristics. Efforts to control leishmaniasis have focused on its diagnosis and treatment. This has led to the implementation of alternative measures targeting sand fly vectors for disease control, based on the antagonistic relationships between some bacteria in the digestive tract of sand flies and parasites of the Leishmania genus. However, their application for leishmaniasis control is limited by a lack of knowledge regarding the frequency with which these interactions occur in nature. Biological control with the bacterium Wolbachia has been successfully used to mitigate viral diseases in mosquitoes carrying the arboviruses, Dengue and Chikungunya. This endosymbiont also interferes with the establishment of Plasmodium apicomplexan. In this scenario, metagenomic tools offer the opportunity to characterize the bacteriome of vectors to establish potential antagonistic relationships that protect insects from parasitic infection. Objective. To characterize bacterial endosymbionts and intestinal microbiota in vectors of Leishmania and other trypanosomatids in the Department of Sucre, Caribbean region of Colombia. Methods. This prospective descriptive study was conducted between November 2021 and February 2023 in the Department of Sucre in the municipalities of Sincelejo, Colosó, Sampués, and Ovejas. Sand flies were collected using active search, Shannon, and CDC light traps. The collected females were kept alive until dissection for taxonomic identification and microscopic search for flagellates in the intestinal tract. RESUMEN.................................................5ABSTRACT...............................................7INTRODUCCIÓN.....................................9MARCO TEÓRICO..................................14Los flebotominos como vectores de Leishmania.............14Los parásitos tripanosomátidos y el género Leishmania.............18Endosimbiontes bacterianos como potenciales biocontroladores de Leishmania.......21Estudios del Microbioma en vectores de Leishmania...........24OBJETIVO GENERAL.........................30OBJETIVOS ESPECÍFICOS.....................30MATERIALES Y MÉTODOS...........................31Tipo y área de estudio....................31Recolección de hembras flebotominas...............................33Disección de flebotominos y búsqueda de tripanosomátidos por microscopía.........34Extracción de ADN..............................34Frecuencia de infección con endosimbiontes bacterianos de hembras flebotominas infectadas naturalmente y no infectadas con tripanosomátidos..35Identificación de genogrupos de endosimbiontes bacterianos detectados en hembras flebotominas naturalmente y no infectadas con tripanosomátido..37Identificación de parásitos tripanosomátidos en intestinos de hembras flebotominas del departamento de Sucre....38Caracterización de la microbiota intestinal de hembras de Pintomyia evansi y Micropygomyia cayennensis cayennensis naturalmente infectadas y no infectadas con tripanosomátidos..39RESULTADOS...............................................42Captura e identificación de hembras flebotominas del departamento de Sucre.....42Infección por parásitos flagelados en intestinos de hembras flebotominas.............44Prevalencia de tripanosomátidos mediante PCR anidada (nPCR) de 18S ribosomal en intestinos de hembras flebotominas.....45Prevalencia de Wolbachia y Cardinium en intestinos de hembras flebotominas....46Identificación de genogrupos de endosimbiontes bacterianos en flebotominos...48Identificación de Leishmania y otros tripanosomátidos en intestinos de hembras flebotominas.....50Caracterización del microbioma intestinal de Pintomyia evansi y Micropygomyia cayennensis cayennensis naturalmente infectadas y no infectadas con tripanosomátidos..53DISCUSIÓN..........................63Flebotominos y su importancia epidemiológica......................63Prevalencia de Wolbachia y Cardinium en infecciones con parásitos tripanosomátidos..................65Genotipificación de endosimbiontes asociados a hembras flebotominas...........67Identificación de Leishmania y otros tripanosomátidos en hembras flebotominas.........69Microbioma bacteriano de Pintomyia evansi y Micropygomyia cayennensis cayennensis.............70CONCLUSIONES.......................75BIBLIOGRAFÍA....................77ANEXOS.............................99DoctoradoDoctor(a) en Microbiología y Salud TropicalTrabajos de Investigación y/o Extensiónapplication/pdfspaUniversidad de CórdobaFacultad de Medicina Veterinaria y ZootecniaBerástegui, Córdoba, ColombiaDoctorado en Microbiología y Salud TropicalCopyright Universidad de Córdoba, 2025https://creativecommons.org/licenses/by-nc-nd/4.0/Atribución-NoComercial-SinDerivadas 4.0 Internacional (CC BY-NC-ND 4.0)info:eu-repo/semantics/embargoedAccesshttp://purl.org/coar/access_right/c_f1cfEndosimbiontes bacterianos y microbiota intestinal de vectores de Leishmania y otros tripanosomátidos del departamento de Sucre, región Caribe de ColombiaTrabajo de grado - Doctoradoinfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/acceptedVersionTexthttp://purl.org/redcol/resource_type/TMAdams, Z., & Shimabukuro, P. 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Series B: Biological Sciences, 265(1395), 509-515. https://doi.org/10.1098/rspb.1998.0324EndosimbiontesVectorFlebotominosAgentes de control biológicoLeishmaniaTrypanosomatidaeSecuenciación MinIONEndosymbiontsVectorPhlebotomine sandfliesBiological control agentsLeishmaniaTrypanosomatidaeMinION sequencingPublicationLICENSElicense.txtlicense.txttext/plain; charset=utf-815543https://repositorio.unicordoba.edu.co/bitstreams/d9218980-c106-4ff8-9ad3-4ff90823e318/download73a5432e0b76442b22b026844140d683MD53ORIGINALAlemánSantosMairaAlejandra.pdfAlemánSantosMairaAlejandra.pdfapplication/pdf10904307https://repositorio.unicordoba.edu.co/bitstreams/9f67a5de-5812-4b41-be1b-eed2b4885232/downloadbcfe0c759fd6a6f0747918f5d289420bMD54Autorización Publicación pdf.pdfAutorización Publicación 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