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Detección Y Estudio Molecular, Biológico, Morfológico Y Evolutivo De La Primera Especie Autóctona De Trichinella Hallada En La Región Neotropical

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Detección Y Estudio Molecular, Biológico, Morfológico Y Evolutivo De La Primera Especie Autóctona De Trichinella Hallada En La Región Neotropical Tesis Doctoral Detección y estudio molecular, biológico, morfológico y evolutivo de la primera especie autóctona de Trichinella hallada en la región Neotropical Krivokapich, Silvio Jesús 2017-10-10 Este documento forma parte de la colección de tesis doctorales y de maestría de la Biblioteca Central Dr. Luis Federico Leloir, disponible en digital.bl.fcen.uba.ar. Su utilización debe ser acompañada por la cita bibliográfica con reconocimiento de la fuente. This document is part of the doctoral theses collection of the Central Library Dr. Luis Federico Leloir, available in digital.bl.fcen.uba.ar. It should be used accompanied by the corresponding citation acknowledging the source. Cita tipo APA: Krivokapich, Silvio Jesús. (2017-10-10). Detección y estudio molecular, biológico, morfológico y evolutivo de la primera especie autóctona de Trichinella hallada en la región Neotropical. Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Cita tipo Chicago: Krivokapich, Silvio Jesús. "Detección y estudio molecular, biológico, morfológico y evolutivo de la primera especie autóctona de Trichinella hallada en la región Neotropical". Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. 2017-10-10. Dirección: Biblioteca Central Dr. Luis F. Leloir, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires. Contacto: [email protected] Intendente Güiraldes 2160 - C1428EGA - Tel. (++54 +11) 4789-9293 UNIVERSIDAD DE BUENOS AIRES Facultad de Ciencias Exactas y Naturales Departamento de Ecología, Genética y Evolución Detección y estudio molecular, biológico, morfológico y evolutivo de la primera especie autóctona de Trichinella hallada en la región Neotropical Tesis presentada para optar al título de Doctor de la Universidad de Buenos Aires en el área de Ciencias Biológicas Lic. Silvio Jesús Krivokapich Directora de tesis: Dra. Viviana Andrea Confalonieri Consejera de estudios: Dra. Viviana Andrea Confalonieri Lugar de trabajo: Departamento de Parasitología, Instituto Nacional de Enfermedades Infecciosas, Administración Nacional de Laboratorios e Institutos de Salud, “Dr. Carlos G. Malbrán” Buenos Aires, 2017 Detección y estudio molecular, biológico, morfológico y evolutivo de la primera especie autóctona de Trichinella hallada en la región Neotropical RESUMEN La trichinellsosis es una zoonosis producida por el consumo de carne cruda o semicruda, derivada de cerdos domésticos o animales silvestres, infectada con larvas musculares del nematode Trichinella. Durante muchos años, se consideró a T. spiralis como la única especie del género en Sudamérica. En el presente trabajo se describen cuatro aislamientos de Trichinella hallados en pumas silvestres (Puma concolor), procedentes de distintas provincias de Argentina (Santa Cruz, Rio Negro, Catamarca), donde el análisis de identificación molecular arrojó un patrón distinto a T. spiralis. Los valores de identidad nucleotídica y distancia-p y, particularmente, el análisis filogenético con marcadores de ADN nuclear y mitocondrial señalan que los cuatro aislamientos pertenecen a un nuevo genotipo denominado Trichinella T12, ya que representa el duodécimo miembro del género. El estudio filogenético multilocus muestra que Trichinella T12 se ubica en posición basal a todas las especies y genotipos encapsulados que se distribuyen en la regiónes Neártica, Paleartica y Africana, excepto T. spiralis y T. nelsoni que forman un grupo hermano, unido basalmente a las restantes especies encapsuladas. El análisis biogeográfico permite proponer una hipótesis sobre la diversificación del género Trichinella entre el Mioceno medio y el Pleistoceno medio. El ingreso de Trichinella en la región Neotropical habría sido conducido por mamíferos carnívoros en el inicio del Plioceno luego de la formación del Istmo de Panamá. Los ensayos de cruzamientos recíprocos revelan que el nuevo genotipo esta aislado reproductivamente de las especies del mismo clado. Los estudios sobre longitud uterina y larviposición, sugieren una baja fecundidad de Trichinella T12. El nuevo genotipo presentó una infectividad inferior en cerdos, ratas y ratones respecto a T. spiralis, en concordancia con la historia biogeográfica de ambos parásitos con esos hospederos omnívoros. Las larvas musculares de Trichinella T12 no mostraron resistencia al congelamiento a -18°C, pero mantuvieron inalterada su infectividad durante un mes a la temperatura de -5°C. Adicionalmente, un estudio serológico en ocho pacientes implicados en el consumo de carne de un puma infectado con Trichinella T12, reveló tres casos positivos. En conjunto, los datos señalan que el genotipo en estudio es una nueva especie ampliamente distribuida en Argentina, autóctona de la región Neotropical, que representa un riesgo directo de infección en humanos, y cuyo nombre propuesto es T. patagoniensis. Palabras Clave: Trichinella patagoniensis, Puma concolor, Neotrópico, infectividad, aislamiento reproductivo, filogenia, biogeografía, evolución. Detection and molecular, biological, morphological and evolutionary study of the first autochthonous Trichinella species found in the Neotropical region ABSTRACT Trichinellosis is a zoonotic disease caused by the ingestion of raw or semi-raw meat of pork and wild animals infected with muscle larvae of the nematode Trichinella. For many years, T. spiralis was considered to be the only species of the genus in South America. In this study, we describe four Trichinella isolates from wild cougars (Puma concolor) caught in different provinces of Argentina (Santa Cruz, Rio Negro and Catamarca). The molecular identification analysis showed a pattern different from that of T. spiralis. The values of nucleotide identity and p-distance and, particularly, phylogenetic analyses of nuclear and mitochondrial DNA markers indicate that the four isolates belong to a new genotype, which was named Trichinella T12, as it is the twelfth member of the genus so far reported. The multilocus phylogenetic study shows that Trichinella T12 is in basal position with respect to all the encapsulated species and genotypes distributed in the Nearctic, Palearctic and African regions, except for T. spiralis and T. nelsoni which form a sister group clustered basally to the remaining encapsulated species. The biogeographic analysis has led to propose a hypothesis of the diversification of the genus Trichinella between the medium Miocene and the medium Pleistocene. Trichinella was possibly introduced into the Neotropical region through carnivorous mammals during the early Pliocene after the rise of the Panama isthmus. The results of reciprocal crosses reveal that the new genotype is reproductively isolated from species of the same clade. Studies on uterus length and larvaposition suggest that the fecundity of Trichinella T12 is low. The infectivity of the new genotype in pigs, rats and mice is lower than that of T. spiralis, in agreement with the biogeographic history of the association between both parasites and their omnivorous hosts. Muscle larvae of Trichinella T12 have no resistance to freezing at -18°C, but their infectivity remains unchanged for 30 d at -5°C. In addition, a serological study performed in eight patients who had eaten meal of a cougar infected with Trichinella T12, revealed three positive cases. Overall, data indicate that the genotype under study is a new species autochthonous to the Neotropical region and widely distributed in Argentina, which poses a direct infection risk to humans. The name T. patagoniensis is proposed for this parasite. Key words: Trichinella patagoniensis, Puma concolor, Neotropic, infectivity, reproductive isolation, phylogeny, biogeography, evolution. AGRADECIMIENTOS A Cinthia Gonzalez Prous y Graciana Gatti por acompañarme desde el inicio en esta aventura de describir y caracterizar una nueva especie autóctona de un parasito de nuestro país. A Viviana Confalonieri, por aceptarme como su doctorando y por brindarme su tiempo y experiencia en la realización de esta tesis. A Hugo Matarasso, Luisa Saldia y Bruno Malandrini por aportar las muestras con los aislamientos de Trichinella que permitieron realizar el presente estudio A Patricia Arbusti y Graciana Ayesa por integrar nuestro grupo de trabajo y en particular por su colaboración en el serodiagnóstico de las muestras de sueros humano provenientes de El Calafate. A Fernanda Degese por su ayuda tanto en la mesada como en la corrección de la tesis. A Edoardo Pozio, Giuseppe La Rosa y Gianluca Marucci por brindarme su colaboración en el análisis de aislamiento reproductivo de Trichinella T12 y sus aportes críticos en la descripción de una nueva especie de Trichinella. A Mabel Ribicich, Fernando Fariña y Mariana Pasqualletti por su colaboración en el estudio de la infectividad en cerdos y resistencia térmica en carnívoros de Trichinella T12. A Marcela Amoroso y Alejandra Oliveira del servicio del Bioterio Central del INPB, por el suministro de animales, ratas y ratones, y el apoyo técnico para la experiencias realizadas en estas tesis. A Eduardo Guarnera y Viviana Molina por darme la oportunidad e incentivarme en trabajar con Trichinella. A todas las personas que integran e integraron el Departamento de Parasitología del Malbrán, que facilitaron la realización de los trabajos que redundaron en esta tesis. A Rocco y Mirko, mis hijos, y a Naty, mi esposa, por permitirme el tiempo para realizar y redactar este trabajo de tesis, y motivarme para culminar con esta tarea. Y en especial a mi mamá que, como dice el tango, se nos fue pero aún me guía. INDICE
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