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Teză De Doctorat UNIVERSITATEA DE ȘTIINȚE AGRICOLE ȘI MEDICINĂ VETERINARĂ „ION IONESCU DE LA BRAD” IAȘI, ROMÂNIA FACULTATEA DE MEDICINĂ VETERINARĂ ȘCOALA DOCTORALĂ DE MEDICINĂ VETERINARĂ DOMENIUL DE DOCTORAT MEDICINĂ VETERINARĂ SPECIALIZAREA BOLI INFECȚIOASE TEZĂ DE DOCTORAT Doctorand, MIHAELA-ANCA DASCĂLU Conducător de doctorat, Prof.univ.dr. ELENA VELESCU 2017 UNIVERSITATEA DE ȘTIINȚE AGRICOLE ȘI MEDICINĂ VETERINARĂ „ION IONESCU DE LA BRAD” IAȘI, ROMÂNIA FACULTATEA DE MEDICINĂ VETERINARĂ ȘCOALA DOCTORALĂ DE MEDICINĂ VETERINARĂ DOMENIUL DE DOCTORAT MEDICINĂ VETERINARĂ SPECIALIZAREA BOLI INFECȚIOASE TEZĂ DE DOCTORAT CERCETĂRI PRIVIND METODOLOGIA DE DIAGNOSTIC, SUPRAVEGHERE, PREVENIRE ȘI CONTROL ÎN RABIE Doctorand, MIHAELA-ANCA DASCĂLU Conducător de doctorat, Prof.univ.dr. ELENA VELESCU 2017 „ ION IONESCU DE LA BRAD” UNIVERSITY OF AGRICULTURE SCIENCES AND VETERINARY MEDICINE OF IAȘI, ROMANIA FACULTY OF VETERINARY MEDICINE DOCTORAL SCHOOL OF VETERINARY MEDICINE DOCTORAL FIELD VETERINARY MEDICINE SPECIALTY INFECTIOUS DISEASES PhD THESIS RESEARCH REGARDING DIAGNOSTIC METHODOLOGY, SURVEILLANCE, PREVENTION AND CONTROL IN RABIES PhD student, MIHAELA-ANCA DASCĂLU PhD coordinator, Prof.univ.dr. ELENA VELESCU 2017 CUPRINS LISTA DE ABREVIERI 9 INTRODUCERE 10 REZUMAT 14 PARTEA I STADIUL ACTUAL AL CUNOAȘTERII CAPITOLUL I PARTICULARITĂȚI PRIVIND ETIOLOGIA ȘI CARACTERELE 29 EPIDEMIOLOGICE ALE INFECȚIEI RABICE LA ANIMALE 1.1 Etiologie 29 1.1.1 Structura și morfologia virusului rabic 29 1.1.2 Proteinele virusului rabic 29 1.1.3 Ciclul de infecție și replicare al virusului rabic 30 1.1.4 Încadrarea genetică 31 1.1.5 Rezistența virusului rabic la factorii fizici și 33 chimici 1.2 Caractere epidemiologice 34 1.2.1 Receptivitate 34 1.2.2 Surse de infecție și căi de transmitere 35 1.2.3 Dinamica bolii 35 CAPITOLUL II ACTUALIZĂRI PRIVIND METODOLOGIA DE DIAGNOSTIC, 39 SUPRAVEGHERE, CONTROL ȘI ERADICARE A RABIEI LA ANIMALE 2.1 Metodologia de diagnostic 39 2.2 Metodologia de supraveghere, control și eradicare a rabiei 44 la animale PARTEA A-II-A CONTRIBUȚII PERSONALE CAPITOLUL III SCOPUL, OBIECTIVELE TEZEI DE DOCTORAT ȘI CADRUL 48 ORGANIZATORIC AL CERCETĂRILOR CAPITOLUL IV INVESTIGAȚII EPIDEMIOLOGICE PRIVIND PREZENȚA ȘI 52 PREVALENȚA INFECȚIEI RABICE LA ANIMALE ÎN NORD-ESTUL ROMÂNIEI ȘI ÎN REPUBLICA MOLDOVA 4.1 Investigații epidemiologice privind prezența și prevalența 52 infecției rabice la animale în nord-estul României 4.2 Investigații epidemiologice privind prezența și prevalența 69 infecției rabice la animale în Republica Moldova CAPITOLUL V CONFIRMAREA DIAGNOSTICULUI DE RABIE PRIN METODA DE 74 IMUNOFLUORESCENȚĂ DIRECTĂ 5.1 Materiale și metode 75 5.2 Rezultate 80 5.3 Discuții 83 5.4 Concluzii parțiale 84 CAPITOLUL VI CONFIRMAREA DIAGNOSTICULUI DE RABIE PRIN TEHNICA 85 DE IZOLARE A VIRUSULUI RABIC PE CULTURI CELULARE (RTCIT) 6.1. Izolarea virusului rabic pe culturi celulare (RTCIT) ca 86 metodă alternativă pentru proba biologică pe șoareci 3 6.1.1 Materiale și metode 86 6.1.2 Rezultate și discuții 89 6.1.3 Concluzii parțiale 90 6.2 Studiu comparativ privind sensibilitatea a două linii 91 celulare distincte N2a și BHK-21 utilizate pentru izolarea virusului rabic pe culturi celulare 6.2.1 Materiale și metode 91 6.2.2 Rezultate și discuții 92 6.2.3 Concluzii parțiale 95 6.3. Utilizarea a două sisteme diferite pentru izolarea virusului 95 rabic pe culturi celulare 6.3.1 Materiale și metode 95 6.3.2 Rezultate 97 6.3.3 Discuții 99 6.3.4 Concluzii parțiale 100 CAPITOLUL VII CONFIRMAREA DIAGNOSTICULUI DE RABIE PRIN TEHNICILE 101 DE BIOLOGIE MOLECULARĂ REAL TIME PCR ȘI RT-PCR CONVENȚIONAL 7.1 Materiale și metode 102 7.1.1 Extracția ARN 103 7.1.2 SYBRGreen Real time qRT-PCR 104 7.1.3 TaqMan Real time RT-PCR 106 7.1.4 Heminested RT-PCR convențional 107 7.1.4.1 Amplificarea genei parțiale N a virusului 108 rabic 7.1.4.2 Amplificarea genei complete N a 109 virusului rabic 7.1.5 Electroforeza: migrarea produșilor PCR în gel de 110 agaroză 2% 7.2 Rezultate și discuții 111 7.2.1 SYBRGreen Real time qRT-PCR 111 7.2.2 TaqMan Real time qRT-PCR 116 7.2.3 Heminested RT-PCR convențional 117 7.2.4 Dozarea concentrației de ARN/ADNc viral 121 7.3 Concluzii parțiale 122 CAPITOLUL VIII CARACTERIZAREA MOLECULARĂ A TULPINILOR DE VIRUS 123 RABIC IZOLATE DIN NORD-ESTUL ROMÂNIEI ȘI DIN REPUBLICA MOLDOVA 8.1 Secvențierea și analiza filogenetică a nucleoproteinei 123 virusului rabic 8.1 Materiale și metode 124 8.2 Rezultate și discuții 125 8.3 Concluzii parțiale 138 CAPITOLUL IX SECVENȚIEREA COMPLETĂ A GENOMULUI VIRUSULUI RABIC 139 9.1 Materiale și metode 139 4 9.2 Rezultate și discuții 142 9.3 Concluzii parțiale 154 CAPITOLUL X CERCETĂRI PRIVIND STRATEGIA DE SUPRAVEGHERE ȘI 155 CONTROL A INFECȚIEI RABICE LA ANIMALE ÎN NORD-ESTUL ROMÂNIEI 10.1. Detecția anticorpilor antirabici postvaccinali și 156 determinarea markerului vaccinal - tetraciclina - la vulpi 10.1.1 Materiale și metode 158 10.1.2 Rezultate 158 10.1.2.2 Detecția anticorpilor antirabici la vulpi 163 prin intermediul metodei serologice ELISA 10.1.2.3 Determinarea markerului vaccinal – 166 tetraciclina - la vulpi 10.1.3 Discuții 167 10.1.4 Concluzii parțiale 167 10.2 Detecția anticorpilor antirabici la mistreți 168 10.2.1 Materiale și metode 169 10.2.2 Rezultate 169 10.2.2.1 Detecția anticorpilor antirabici la 172 mistreți prin intermediul testului ELISA 10.2.2.2 Detecția anticorpilor antirabici la 155 mistreți prin intermediul metodei de referință FAVN 10.2.3 Discuții 156 10.2.4 Concluzii parțiale 158 CAPITOLUL XI CONCLUZII FINALE 176 BIBLIOGRAFIE 181 LISTA DE FIGURI 192 LISTA DE TABELE 196 LISTA DE LUCRĂRI 198 5 CONTENTS LIST OF ABBREVIATIONS 9 INTRODUCTION 10 ABSTRACT 14 FIRST PART CURRENT STATE OF KNOWLEDGE CHAPTER I PARTICULARITIES ON THE ETIOLOGY AND EPIDEMIOLOGICAL 29 CHARACTERISTICS OF THE RABIES INFECTION IN ANIMALS 1.1 Etiology 29 1.1.1 Structure and morfology of rabies virus 29 1.1.2 Rabies virus proteins 29 1.1.3 Cycle of infection and replication of rabies virus 30 1.1.4 Genetic framing 31 1.1.5 Resistance of rabies virus to physical and 33 chemical factors 1.2 Epidemiology 34 1.2.1 Receptivity 34 1.2.2 Sources of infection and transmission pathways 35 1.2.3 Disease dynamics 35 CHAPTER II UPDATES REGARDING RABIES DIAGNOSTIC METHODOLOGY, 39 SURVEILLANCE, CONTROL AND ERADICATION IN ANIMALS 2.1 Diagnostic methodology 39 2.2 Surveillance, control and eradication methodology 44 of rabies in animals SECOND PART PERSONAL CONTRIBUTION CHAPTER III PhD THESIS AIM AND OBJECTIVES AND THE ORGANIZATIONAL 48 FRAMEWORK OF THE RESEARCHES CHAPTER IV EPIDEMIOLOGICAL INVESTIGATIONS REGARDING THE 52 PRESENCE AND PREVALENCE OF RABIES INFECTION IN ANIMALS IN NORTH-EAST OF ROMANIA AND REPUBLIC OF MOLDOVA 4.1 Epidemiological investigations regarding the presence and 52 prevalence of rabies infection in animals in north-east of Romania 4.2 Epidemiological investigations regarding the presence and 69 prevalence of rabies infection in animals in Republic of Moldova CHAPTER V RABIES DIAGNOSTIC CONFIRMATION BY DIRECT 74 FLUORESCENT ANTIBODY TEST 5.1 Materials and methods 75 5.2 Results 80 5.3 Discussions 83 5.4 Partial conclusions 84 CHAPTER VI RABIES DIAGNOSTIC CONFIRMATION BY RABIES TISSUE 85 CULTURE INFECTION TEST 6 6.1. Isolation of rabies virus on cell cultures (RTCIT) as 86 alternative to the Mouse Inoculation Test 6.1.1 Materials and methods 86 6.1.2 Results and discussions 89 6.1.3 Partial conclusions 90 6.2 Comparative study regarding the sensitivity of two 91 different cell lines used for isolation of rabies virus on cell cultures 6.2.1 Materials and methods 91 6.2.2 Results and discussions 92 6.2.3 Partial conclusions 95 6.3. The use of two different systems for the isolation of rabies 95 virus on cell cultures 6.3.1 Materials and methods 95 6.3.2 Results 97 6.3.3 Discussions 99 6.3.4 Partial conclusions 100 CHAPTER VII RABIES DIAGNOSTIC CONFIRMATION BY REAL TIME PCR AND 101 CONVENTIONAL RT-PCR TECHNIQUES 7.1 Materials and methods 102 7.1.1 RNA extraction 103 7.1.2 SYBRGreen Real time qRT-PCR 104 7.1.3 TaqMan Real time RT-PCR 106 7.1.4 Heminested conventional RT-PCR 107 7.1.4.1 Partial N gene amplification of rabies 108 virus 7.1.4.2 Full N gene amplification of rabies virus 109 7.1.5 Electrophoresis: migration of PCR products into 110 2% agarose gel 7.2 Results and discussions 111 7.2.1 SYBRGreen Real time qRT-PCR 111 7.2.2 TaqMan Real time qRT-PCR 116 7.2.3 Heminested conventional RT-PCR 117 7.2.4 cDNA/RNA dosage concentrations 121 7.3 Partial conclusions 122 CHAPTER VIII MOLECULAR CHARACTERIZATION OF RABIES VIRUS 123 STRAINS FROM NORTH-EAST OF ROMANIA AND REPUBLIC OF MOLDOVA 8.1 Sequencing and phylogenetic analysis of rabies virus 123 nucleoproteine 8.1 Materials and methods 124 8.2 Results and discussions 125 8.3 Partial conclusions 138 CHAPTER IX THE COMPLETE SEQUENCING OF RABIES VIRUS FULL 139 GENOME 7 9.1 Materials and methods 139 9.2 Results and discussions 142 9.3 Partial conclusions 154 CHAPTER X RESEARCHES REGARDING THE SURVEILANCE AND CONTROL 155 STRATEGY OF ANIMALS RABIES INFECTION IN THE NORTH- EAST OF ROMANIA 10.1. Detection of antirabies antibodies and determinations of 155 tetracycline in foxes 10.1.1 Materials and methods 156 10.1.2 Results 158 10.1.2.2 Detection of antirabies antibodies in 158 foxes by ELISA 10.1.2.3 Detections of tetracycline in foxes 163 mandibles 10.1.3 Discussions 166 10.1.4 Partial conclusions 167 10.2 Detections of antirabies antibodies in wild boars 167 10.2.1 Materials and methods 168 10.2.2 Results 169 10.2.2.1 Detections of antirabies antibodies in 169 wild boar by ELISA 10.2.2.2 Detections
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