UNIVERSIDAD DE CÓRDOBA FACULTAD DE VETERINARIA TESIS DOCTORAL Dña. Verónica ...

194 Pages·2013·9.91 MB·English

UNIVERSIDAD DE CÓRDOBA
FACULTAD DE VETERINARIA

TESIS DOCTORAL

SYSTEMIC AND LOCAL CELL-MEDIATED IMMUNE RESPONSE AGAINST BHV-1
IN HEALTHY AND BVDV-INFECTED CALVES

RESPUESTA INMUNE CELULAR SISTÉMICA Y LOCAL FRENTE AL HVB-1
EN TERNEROS SANOS Y TERNEROS CON EL vDVB

Dña. Verónica María Molina Hernández
Departamento de Anatomía y Anatomía Patológica Comparadas
Córdoba, Marzo 2013
TITULO: RESPUESTA INMUNE CELULAR SISTÉMICA Y LOCAL FRENTE AL
HVB-1 EN TERNER0OS SANOS Y TERNEROS CON vDVB.
AUTOR: VERÓNICA MARÍA MOLINA HERNÁNDEZ
© Edita: Servicio de Publicaciones de la Universidad de Córdoba.
Campus de Rabanales
Ctra. Nacional IV, Km. 396 A
14071 Córdoba
www.uco.es/publicaciones
[email protected]
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A mi familia
A Pedro

A todos los que iluminan mi camino
“Casi
 todos
 los
 que
 desconfían
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ignoran
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 prolongada”

De
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D. Santiago Ramón y Cajal
TABLE OF CONTENTS
TABLE OF CONTENTS

ABBREVIATIONS ................................................................................................................... 11

PREFACE: Aim and Scope of the thesis ................................................................................ 15

GENERAL INTRODUCTION .................................................................................................... 18
I. Bovine Viral Diarrhea Virus (BVDV) ........................................................................... 19
1. History of Bovine Viral Diarrhea (BVD) .......................................................... 19
2. Classification, Morphology and Structure of BVDV ....................................... 20
3. Clinical Manifestations of BVDV Infections in Cattle ..................................... 24
3.1. Acute BVD ............................................................................................ 25
Severe acute BVD .......................................................................... 26
Hemorrhagic .................................................................................. 27
3.2. Congenital persistent infection ........................................................... 27
3.3. Mucosal disease (MD) ......................................................................... 28
4. Pathogenesis of Acute BVD ............................................................................ 29
4.1. Target cell and Spread of BVDV........................................................... 29
4.2. BVDV and Immunity ............................................................................ 32
4.2.1. Immunosuppression ................................................................. 33
4.2.2. Innate Immunity ....................................................................... 33
4.2.3. Adaptive Immunity ................................................................... 34
4.2.3.1. Humoral immune response ........................................ 35
4.2.3.2. Cell-mediated immune response ............................... 35
4.2.4. Role of Cytokines in BVD .......................................................... 38
4.3. BVDV and Concurrent Infections ......................................................... 40

Verónica María Molina Hernández 6
TABLE OF CONTENTS
II. Bovine Herpesvirus Type 1 (BHV-1) ......................................................................... 42
1. History, Classification and Clinical Manifestations ........................................ 42
2. Pathogenesis .................................................................................................. 45
3. BHV-1 and Immunity ...................................................................................... 47
3.1. Immunosuppression ............................................................................ 48
3.2. Humoral Immune Response ................................................................ 48
3.3. Cell-mediated Immune Response ....................................................... 49

III. Nasal mucosa and palatine tonsil ............................................................................ 50
1. Mucosa-Associated Lymphoid Tissue (MALT) ............................................... 50
2.  Composition  of  the  Bovine  Waldeyer’s  ring .................................................. 51
3. Tonsillar Structures ........................................................................................ 51
3.1. Tonsillar Epithelium ............................................................................. 52
3.2. Lymphoepithelium .............................................................................. 53
4. Role of tonsils in infection .............................................................................. 54

OBJECTIVES .......................................................................................................................... 56

MATERIALS AND METHODS ................................................................................................. 58
1. Experimental outline ...................................................................................... 59
2. Samples collection ......................................................................................... 61
2.1. Blood, serum and plasma samples ...................................................... 61
2.2. Tissue samples ..................................................................................... 62
3. Methods and techniques ............................................................................... 64
3.1. Viruses detection by PCR or virological examination .......................... 64
3.2. Antibodies detection ........................................................................... 65
3.3. Total and differential leukocyte counts .............................................. 65
3.4. Flow cytometric analysis of lymphocyte subpopulations ................... 65
3.5. Acute phase protein assays ................................................................. 67
3.6. Cytokine assays ................................................................................... 68

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TABLE OF CONTENTS
4. Histology and immunohistochemical study ................................................... 69
4.1. Evaluation of immunostaining ............................................................ 70
5. Statistical analyses ......................................................................................... 71

CHAPTER 1: Cell-mediated immune response during experimental acute infection with
bovine viral diarrhea virus (BVDV): evaluation of blood parameters .................................. 72
Abstract .............................................................................................................. 73
1. Introduction ................................................................................................... 74
2. Materials and Methods .................................................................................. 76
2.1. Experimental procedure ................................................................ 76
2.2 Virus and specific antibody detection ............................................. 77
2.3. Leukocyte counts and lymphocyte subset analyses ...................... 78
2.4. APP assays ...................................................................................... 79
2.5. Cytokine assays .............................................................................. 79
2.6. Statistics ......................................................................................... 80
3. Results ............................................................................................................ 80
3.1. Assessment of clinical symptoms ................................................... 80
3.2. Viremia and anti-BVDV antibodies ................................................. 81
3.3. Leukocyte populations ................................................................... 82
3.4. Lymphocyte subpopulations .......................................................... 84
3.5. APP analysis .................................................................................... 85
3.6. Cytokine analysis ............................................................................ 85
3.6.1. Proinflammatory cytokines ................................................. 87
3.6.2. Type 1 and type 2 cytokines ................................................ 87
4. Discussion ....................................................................................................... 89

CHAPTER 2: Effect of infection with BHV-1 on peripheral blood leukocytes and lymphocyte
subpopulations in calves with subclinical BVD .................................................................... 94
Abstract .............................................................................................................. 95
1. Introduction ................................................................................................... 96
2. Materials and Methods .................................................................................. 97

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