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Estudio De Microorganismos De La Clase Mollicutes En Organismos Marinos

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Estudio De Microorganismos De La Clase Mollicutes En Organismos Marinos UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA FACULTAD DE VETERINARIA INSTITUTO UNIVERSITARIO DE SANIDAD ANIMAL Y SEGURIDAD ALIMENTARIA TESIS DOCTORAL Estudio de microorganismos de la Clase Mollicutes en organismos marinos. Orestes Manuel Vega-Orellana LAS PALMAS DE GRAN CANARIA 2014 I. APITULO I I. Índices II. ÍNDICE GENERAL I . ÍNDICE GENERAL .................................................................................................................. I II . ÍNDICE DE TABLAS ............................................................................................................. VII III. ÍNDICE DE FIGURAS ............................................................................................................. X IV. AGRADECIMIENTOS .......................................................................................................... XV V. RESUMEN ...................................................................................................................... XVII VI. ABSTRACT......................................................................................................................... XIX VII.ABREVIATURAS ................................................................................................................ XXI I. CAPÍTULO I: Introducción, Objetivos y Revisión Bibliográfica ......................................... 1 I.1 INTRODUCCIÓN.............................................................................................................. 1 I.2 OBJETIVOS ..................................................................................................................... 4 I.2.1 OBJETIVO GENERAL .............................................................................................. 4 I.2.2 OBJETIVOS ESPECÍFICOS...................................................................................... 4 I.3 REVISIÓN BIBLIOGRÁFICA ............................................................................................. 5 I.3.1 MOLLICUTES ........................................................................................................... 5 I.3.1.1 Características generales .................................................................................... 5 I.3.1.2 Taxonomía y filogenia ......................................................................................... 7 I.3.1.3 Características ultraestructurales ........................................................................13 I.3.1.3.1 Estructura celular y cápsula ..........................................................................14 I.3.1.3.2 Membrana celular y estructura antigénica ......................................................15 I.3.2 TÉCNICAS DE IDENTIFICACIÓN Y CARACTERIZACIÓN DE MOLLICUTES ...........16 I.3.2.1 Procedimientos preliminares (Tully y Whitcomb, 1995; Ramírez, 1999) ................16 I.3.2.1.1 Cultivo puro ..................................................................................................16 I.3.2.1.2 Ausencia de pared celular .............................................................................17 I.3.2.1.3 Detección de formas L bacterianas ................................................................17 I.3.2.1.4 Morfología de las colonias .............................................................................17 I.3.2.1.5 Requerimientos de esteroles .........................................................................18 I.3.2.2 Métodos bioquímicos y enzimáticos ....................................................................18 I.3.2.2.1 Hidrólisis de la urea ......................................................................................19 I.3.2.2.2 Hidrólisis de azúcares ...................................................................................19 I.3.2.2.3 Hidrólisis de la arginina .................................................................................22 I.3.2.2.4 Actividad de la fosfatasa ...............................................................................23 i Índices I.3.2.2.5 Reducción del Tetrazolium ........................................................................... 23 I.3.2.2.6 Formación de películas y cristales ................................................................ 24 I.3.2.3 Perfiles de proteínas totales ............................................................................... 25 I.3.2.4 Métodos serológicos .......................................................................................... 25 I.3.2.4.1 Inhibición de crecimiento .............................................................................. 25 I.3.2.4.2 Inhibición del metabolismo ........................................................................... 25 I.3.2.5 Inmunofluorescencia ......................................................................................... 26 I.3.2.6 Inmunohistoquímica .......................................................................................... 27 I.3.2.7 Métodos de biología molecular .......................................................................... 27 I.3.2.7.1 Reacción en Cadena de la Polimerasa – PCR .............................................. 27 I.3.2.7.2 Secuenciación de ADN ................................................................................ 28 I.3.2.7.3 PCR a tiempo real ........................................................................................ 30 I.3.2.7.4 DGGE.......................................................................................................... 30 I.3.2.7.5 Microarrays .................................................................................................. 31 I.3.2.7.6 Tipificación .................................................................................................. 32 I.3.3 HÁBITATS Y ENFERMEDADES.............................................................................. 36 I.3.3.1 Patogenicidad y factores de virulencia .......................................................... 39 I.3.3.2 Enfermedades más importantes producidas por mollicutes ............................ 40 I.3.4 MOLLICUTES Y ORGANISMOS ACUÁTICOS ........................................................... 45 I.3.4.1 Invertebrados .................................................................................................... 45 I.3.4.2 Peces ............................................................................................................... 50 I.3.4.2.1 Mycoplasma mobile ..................................................................................... 53 I.3.4.3 Mamíferos marinos ............................................................................................ 58 II. CAPITULO II: Estudio metodológico – Evaluación de la sensibilidad y especificidad de seis pares de cebadores utilizados en la detección e identificación de microorganismos de la Clase Mollicutes ................................................................................................................. 65 II.1 ANTECEDENTES ........................................................................................................... 65 II.2 MATERIALES Y MÉTODOS ............................................................................................ 70 II.2.1 ORGANISMOS Y CONDICIONES DE CULTIVO ........................................................ 70 II.2.2 EXTRACCIÓN Y MEDICIÓN DE ADN ........................................................................ 70 II.2.3 CONFIRMACIÓN DE LA IDENTIDAD DEL ADN ........................................................ 74 II.2.4 COMPARACIÓN DE SEIS PARES DE CEBADORES (PCR’S). .................................. 75 II.2.5 PRUEBAS DE ESPECIFICIDAD Y SENSIBILIDAD DE LAS PCR’S ............................ 75 ii Índices II.2.5.1 Pruebas de especificidad....................................................................................75 II.2.5.2 Pruebas de sensibilidad .....................................................................................76 II.2.6 PCR A TIEMPO REAL ...............................................................................................77 II.3 RESULTADOS ................................................................................................................78 II.3.1 ORGANISMOS Y ADN ...............................................................................................78 II.3.2 ESPECIFICIDAD........................................................................................................78 II.3.2.1 Mollicutes ..........................................................................................................78 II.3.2.2 Bacterias no-mollicutes ......................................................................................79 II.3.3 PRUEBAS DE SENSIBILIDAD ...................................................................................81 II.3.4 CÁLCULO DE SENSIBILIDAD Y ESPECIFICIDAD .....................................................82 II.4 DISCUSIÓN ....................................................................................................................84 II.4.1 CEBADORES .. ………………………………………………………………………………...85 II.4.2 ESPECIES UTILIZADAS. ...........................................................................................87 II.4.3 MOLLICUTES……… ..................................................................................................89 II.4.4 BACTERIAS NO-MOLLICUTES .................................................................................92 II.4.5
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