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Trobicúlidos Y Trombiculiasis En La Rioja

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Trobicúlidos Y Trombiculiasis En La Rioja TESIS DOCTORAL Título Trobicúlidos y trombiculiasis en La Rioja Autor/es Paula Santibáñez Sáenz Director/es José Antonio Oteo Revuelta y Aránzazu Portillo Barrio Facultad Facultad de Ciencias, Estudios Agroalimentarios e Informática Titulación Departamento Agricultura y Alimentación Curso Académico 2014-2015 Trobicúlidos y trombiculiasis en La Rioja, tesis doctoral de Paula Santibáñez Sáenz, dirigida por José Antonio Oteo Revuelta y Aránzazu Portillo Barrio (publicada por la Universidad de La Rioja), se difunde bajo una Licencia Creative Commons Reconocimiento-NoComercial-SinObraDerivada 3.0 Unported. Permisos que vayan más allá de lo cubierto por esta licencia pueden solicitarse a los titulares del copyright. © El autor © Universidad de La Rioja, Servicio de Publicaciones, 2015 publicaciones.unirioja.es E-mail: [email protected] TESIS DOCTORAL Trombicúlidos y trombiculiasis en La Rioja Trombiculid mites and trombiculiasis in La Rioja Memoria presentada para aspirar al título de Doctor con la Mención de “Doctor Internacional” por la Universidad de La Rioja Paula Santibáñez Sáenz 2015 Don José Antonio Oteo Revuelta, Doctor en Medicina y Cirugía, y Jefe del Departamento de Enfermedades Infecciosas del Hospital San Pedro - Centro de Investigación Biomédica de La Rioja (CIBIR). Doña Aránzazu Portillo Barrio, Doctora en Ciencias Biológicas, y Responsable del Laboratorio de Patógenos Especiales del Departamento de Enfermedades Infecciosas del Centro de Investigación Biomédica de La Rioja (CIBIR). Por la presente declaran que: La memoria titulada “Trombicúlidos y Trombiculiasis en La Rioja”, que presenta Dña. Paula Santibáñez Sáenz, Licenciada en Ciencias Biológicas y en Bioquímica, ha sido realizada en el Centro de Investigación Biomédica de La Rioja (CIBIR) bajo su dirección y reúne las condiciones específicas para optar al grado de Doctor con la mención de “Doctor Internacional”. Lo que hacen constar en Logroño, a 4 de junio de 2015. Fdo.: Dr. José A. Oteo Revuelta Fdo.: Dra. Aránzazu Portillo Barrio ÍNDICE Índice Abreviaturas ........................................................................................................... i Tablas .................................................................................................................... iii Figuras .................................................................................................................... v RESUMEN ............................................................................................................ ix INTRODUCCIÓN ................................................................................................ 1 1. UN POCO DE HISTORIA ................................................................................. 3 2. LOS TROMBICÚLIDOS ................................................................................... 4 2.1. CICLO BIOLÓGICO Y MORFOLOGÍA ................................................................... 4 2.2. DISTRIBUCIÓN ......................................................................................................... 9 2.3. PROCESO DE ALIMENTACIÓN DE LAS LARVAS ............................................ 13 3. LOS TROMBICÚLIDOS COMO PARÁSITOS DE ANIMALES ................. 17 4. LOS TROMBICÚLIDOS COMO VECTORES DE ENFERMEDADES ....... 18 4.1. TIFUS DE LOS MATORRALES.............................................................................. 18 4.2. OTRAS ENFERMEDADES INFECCIOSAS ASOCIADAS A LOS TROMBICÚLIDOS .......................................................................................................... 22 5. LOS TROMBICÚLIDOS Y LA DERMATITIS .............................................. 24 6. LOS TROMBICÚLIDOS EN ESPAÑA .......................................................... 29 OBJETIVOS ........................................................................................................ 31 MATERIAL Y MÉTODOS ............................................................................... 35 1. RECOGIDA Y PREPARACIÓN DE MUESTRAS ......................................... 37 1.1 MUESTREOS DE LARVAS DE TROMBICÚLIDOS DE LA VEGETACIÓN ...... 37 1.1.1. ÉPOCA Y LUGAR ................................................................................................. 37 1.1.2. TÉCNICAS DE RECOGIDA ................................................................................. 39 1.1.2.1. Arrastre ................................................................................................................ 39 1.1.2.2. Captura directa ..................................................................................................... 41 1.1.3. PREPARACIÓN DE LAS MUESTRAS ................................................................ 42 1.2. MUESTREOS DE LARVAS DE TROMBICÚLIDOS EN AVES ........................... 43 1.2.1. ÉPOCA Y LUGAR ................................................................................................. 43 1.2.2. TÉCNICA DE RECOGIDA ................................................................................... 43 1.2.3. PREPARACIÓN DE LAS MUESTRAS ................................................................ 44 1.3. MUESTREOS DE ESTADIOS POST-LARVARIOS DE TROMBICÚLIDOS EN EL SUELO ........................................................................................................................ 44 Índice 1.3.1. ÉPOCA Y LUGAR ................................................................................................. 44 1.3.2. TÉCNICA DE RECOGIDA Y EXTRACCIÓN DE LA EDAFOFAUNA ............ 45 1.3.3. PREPARACIÓN DE LAS MUESTRAS ................................................................ 45 2.1. LARVAS DE TROMBICÚLIDOS ............................................................................ 47 2.2. ESTADIOS POST-LARVARIOS DE TROMBICÚLIDOS...................................... 47 3. TÉCNICAS DE BIOLOGÍA MOLECULAR ................................................... 48 3.1. EXTRACCIÓN DE ADN .......................................................................................... 48 3.2. CUANTIFICACIÓN DE ADN .................................................................................. 49 3.3. REACCIÓN EN CADENA DE LA POLIMERASA (PCR) ..................................... 49 3.3.1. IDENTIFICACIÓN GENÉTICA DE LARVAS DE TROMBICÚLIDOS ............ 51 3.3.2. DETECCIÓN DE BACTERIAS EN LARVAS DE TROMBICÚLIDOS ............. 54 3.3.2.1. ARNr 16S universal de bacterias ......................................................................... 54 3.3.2.2. Rickettsia spp. ...................................................................................................... 54 3.3.2.3. Anaplasma phagocytophilum ............................................................................... 56 3.3.2.4. Borrelia spp. ......................................................................................................... 57 3.3.2.5. Coxiella burnetii ................................................................................................... 58 3.3.2.6. Bartonella spp. ..................................................................................................... 59 3.3.2.7. Orientia tsutsugamushi ........................................................................................ 60 3.4. ELECTROFORESIS EN GEL DE AGAROSA ........................................................ 61 3.5. SECUENCIACIÓN Y ANÁLISIS DE RESULTADOS POSITIVOS ...................... 61 3.6. INCORPORACIÓN DE SECUENCIAS A LA BASE DE DATOS GENBANK ..... 62 4. DESCRIPCIÓN DE CASOS HUMANOS Y CANINOS ................................. 63 4.1. CASOS HUMANOS .................................................................................................. 63 4.2. CASOS CANINOS .................................................................................................... 64 RESULTADOS .................................................................................................... 65 1. RECOGIDA Y PREPARACIÓN DE MUESTRAS ......................................... 67 1.1. MUESTREOS DE LARVAS DE TROMBICÚLIDOS EN VEGETACIÓN ............ 67 1.1.1. AÑO 2008 ............................................................................................................... 69 1.1.2. AÑO 2009 ............................................................................................................... 71 1.1.3. AÑO 2010 ............................................................................................................... 72 1.2. MUESTREOS DE LARVAS DE TROMBICÚLIDOS EN AVES ........................... 77 1.3. MUESTREOS DE ESTADIOS POST-LARVARIOS DE TROMBICÚLIDOS EN EL SUELO ........................................................................................................................ 79 2. CLASIFICACIÓN MORFOLÓGICA .............................................................. 80 2.1 LARVAS DE TROMBICÚLIDOS ............................................................................. 80 Índice 2.1.1. LARVAS RECOGIDAS DE LA VEGETACIÓN ................................................. 80 2.1.2. LARVAS RETIRADAS DE AVES ....................................................................... 82 2.2. ESTADIOS POST-LARVARIOS DE TROMBICÚLIDOS ..................................... 85 3. TÉCNICAS DE BIOLOGÍA MOLECULAR ................................................... 86 3.1 IDENTIFICACIÓN GENÉTICA DE LARVAS DE TROMBICÚLIDOS ................ 86 3.1.1. LARVAS RECOGIDAS DE LA VEGETACIÓN ................................................. 86 3.1.2. LARVAS
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