ADVERTIMENT. Lʼaccés als continguts dʼaquesta tesi queda condicionat a lʼacceptació de les condicions dʼús establertes per la següent llicència Creative Commons: http://cat.creativecommons.org/?page_id=184 ADVERTENCIA. El acceso a los contenidos de esta tesis queda condicionado a la aceptación de las condiciones de uso establecidas por la siguiente licencia Creative Commons: http://es.creativecommons.org/blog/licencias/ WARNING. The access to the contents of this doctoral thesis it is limited to the acceptance of the use conditions set by the following Creative Commons license: https://creativecommons.org/licenses/?lang=en Immune and protective role of vaccines against Salmonella Enteritidis and Salmonella Typhimurium in laying hens Lourdes Cecilia Vañó Sempere PhD Thesis Bellaterra, 2020 Immune and protective role of vaccines against Salmonella Enteritidis and Salmonella Typhimurium in laying hens Tesi doctoral presentada per Lourdes Cecilia Vañó Sempere per accedir al grau de Doctora en el marc del programa de doctorat en Medicina i Sanitat Animals de la Facultat de Veterinària de la Universitat Autònoma de Barcelona, sota la direcció del Doctor Ignacio Badiola Sáiz i la tutoria del Doctor Joaquim Segalés Coma. Bellaterra, 2020 El Doctor Ignacio Badiola Sáiz, investigador de l’Institut de Recerca i Tecnologia Agroalimentàries - Centre de Recerca en Sanitat Animal (IRTA-CReSA) i el Doctor Joaquim Segalés Coma, professor titular del Departament de Sanitat i d’Anatomia Animals de la Facultat de Veterinària de la Universitat Autònoma de Barcelona i investigador adscrit a l’IRTA-CReSA, Certifiquen: Que la memòria titulada “Immune and protective role of vaccines against Salmonella Enteritidis and Salmonella Typhimurium in laying hens”, presentada per Lourdes Cecilia Vañó Sempere per a l’obtenció del grau de Doctora en Medicina i Sanitat Animals, s’ha realitzat sota la seva direcció i tutoria, i n’autoritzen la seva presentació a fi de ser avaluada per la comissió corresponent. I perquè així consti i tingui els efectes que corresponguin, signen el present certificat. Bellaterra (Barcelona), 17 de febrer de 2020 Dr. Ignacio Badiola Sáiz Dr. Joaquim Segalés Coma Lourdes Cecilia Vañó Sempere Director Tutor Doctoranda Signat digitalment Firmado digitalmente por SEGALES Firmado digitalmente JOAQUIM - 33944020E LOURDES per Ignasi Badiola SEGALES Nombre de reconocimiento (DN): por LOURDES CECILIA Saiz - DNI c=ES, CECILIA VAÑO VAÑO SEMPERE - DNI serialNumber=IDCES-33944020E, 48602347N 38540699J (SIG) JOAQUIM - givenName=JOAQUIM, SEMPERE - DNI Fecha: 2020.02.17 Data: 2020.02.17 sn=SEGALES, cn=SEGALES JOAQUIM - 33944020E 48602347N 07:17:44 +01'00' 33944020E 08:30:35 +01'00' Fecha: 2020.02.17 07:39:04 +01'00' La present tesi doctoral ha estat realitzada amb el suport de la Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya (Projecte de Doctorat Industrial, 2015 DI 002) i l’empresa Laboratoris Calier, SA (Barcelona). La impressió d’aquesta tesi ha estat finançada pel programa de doctorat en Medicina i Sanitat Animals de la Facultat de Veterinària de la Universitat Autònoma de Barcelona. Il·lustració de la portada: Mere Sempere Disseny de la portada: Ferran Sempere Table of contents Table of contents .......................................................................................................... I List of abbreviations .................................................................................................. V Abstract ..................................................................................................................... VII Resum ......................................................................................................................... IX Resumen .................................................................................................................... XI 1. GENERAL INTRODUCTION ........................................................................................ 1 1.1 A BRIEF OVERVIEW: ROLE OF Salmonella AS A ZOONOTIC AGENT........... 3 1.1.1 Zoonotic diseases ................................................................................................ 3 1.1.2 The Salmonella status in the European Union ................................................. 4 1.1.3 The Salmonella National Control Programmes ............................................... 6 1.1.4 The use of vaccines for the control of Salmonella ........................................... 7 1.2 THE GENUS Salmonella ............................................................................................. 8 1.2.1 History of Salmonella .......................................................................................... 8 1.2.2 Taxonomy and description ............................................................................... 8 1.2.3 Serotyping of Salmonella genus ....................................................................... 10 1.2.4 Host adaptation of Salmonella serovars ......................................................... 11 1.2.5 Salmonella virulence factors ............................................................................. 12 1.2.5.1 Salmonella Pathogenicity Islands (SPI) ....................................................... 12 1.2.5.2 Surface structures of Salmonella .................................................................. 13 1.2.5.3 Capsular antigen ........................................................................................... 14 1.2.5.4 Lipopolisaccharide (LPS) ............................................................................. 14 1.2.5.5 Fimbriae ......................................................................................................... 15 1.2.5.6 Flagella ........................................................................................................... 15 1.3 THE AVIAN ENTERIC IMMUNE SYSTEM ........................................................ 17 1.4 THE AVIAN IMMUNE RESPONSE TO Salmonella ............................................ 19 1.4.1 Innate immune response ................................................................................. 20 1.4.2 Adaptive humoral immune response ............................................................ 21 1.4.3 Adaptive cellular immune response .............................................................. 22 1.5 Salmonella INFECTION IN CHICKENS ................................................................ 24 1.5.1 Origins of infection ........................................................................................... 24 1.5.2 Mechanisms of Salmonella infection ............................................................... 24 I 1.5.3 Infection with adapted Salmonella serovars .................................................. 26 1.5.4 Infection with non-adapted Salmonella serovars .......................................... 28 1.5.4.1 Salmonella infection in young chicks .......................................................... 28 1.5.4.2 Salmonella infection in adult chickens ........................................................ 28 1.5.5 Contamination of eggs by Salmonella ............................................................. 29 1.6 VACCINES AGAINST Salmonella IN POULTRY PRODUCTION .................... 30 1.6.1 Live attenuated vaccines ................................................................................. 34 1.6.1.1 Metabolic drift mutants ............................................................................... 35 1.6.1.2 Competitive exclusion ................................................................................. 35 1.6.2 Killed vaccines .................................................................................................. 36 1.6.3 Subunit vaccines ............................................................................................... 37 1.6.4 DNA vaccines ................................................................................................... 37 1.6.5 Cross-protection in Salmonella vaccines ........................................................ 37 1.6.6 Regulations for Salmonella vaccines in Europe ............................................. 39 2. HYPOTHESIS AND OBJECTIVES ............................................................................. 41 3. STUDY I: Immunogenicity test of Salmonella Typhimurium vaccine after first vaccination by oral route and spray route in SPF chicks ................................................. 45 3.1 INTRODUCTION ..................................................................................................... 47 3.2 MATERIALS AND METHODS .............................................................................. 48 3.2.1 Animals .............................................................................................................. 48 3.2.2 Bacterial strains ................................................................................................. 49 3.2.3 Study design ...................................................................................................... 49 3.2.4 Detection of Salmonella spp. strains by culture ............................................ 50 3.2.5 Enzyme-linked immunosorbent assay (ELISA) ........................................... 51 3.2.6 Flow cytometry ................................................................................................. 51 3.2.7 Reverse transcriptase quantitative real-time PCR (RT-qPCR) ..................