Submitted by ALINE EYKO KAWANAMI null (aline.kawanami@gmail.com) on 2017-01-12T01:13:37Z No. of bitstreams: 1 Tese_ Aline Kawanami.pdf: 4217654 bytes, checksum: 17280c9aecebf3ab6853cdc956889fb3 (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-01-12T18:53:10Z (GMT) No. of bitstreams: 1 kawanami_ae_dr_jabo.pdf: 4217654 bytes, checksum: 17280c9aecebf3ab6853cdc956889fb3 (MD5) / Made available in DSpace on 2017-01-12T18:53:10Z (GMT). No. of bitstreams: 1 kawanami_ae_dr_jabo.pdf: 4217654 bytes, checksum: 17280c9aecebf3ab6853cdc956889fb3 (MD5) Previous issue date: 2016-12-09 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Língua azul (LA) é uma doença viral infecciosa que afeta ruminantes domésticos e selvagens e é transmitida por mosquitos vetores do gênero Culicoides (Diptera: Ceratopogonidae). É de notificação obrigatória segundo lista da OIE (Organização Mundial de Saúde Animal) e do Ministério da Agricultura, Pecuária e Abastecimento do Brasil. Os sinais clínicos e lesões em casos agudos de infecção pelo vírus da língua azul (VLA) são sutis ou inexistentes; e quando presentes são muito semelhantes com outras enfermidades hemorrágicas, havendo a necessidade de técnicas laboratoriais complementares para o diagnóstico definitivo dessa enfermidade. O presente trabalho teve como objetivo realizar um estudo epidemiológico da doença da língua azul nos cervídeos pertencentes ao Núcleo de Pesquisa e Conservação de Cervídeos – NUPECCE e nos ruminantes domésticos mantidos nas proximidades, utilizando técnicas sorológicas (ELISA competitivo em fase sólida e imunodifusão dupla em gel de ágar - IDGA) e moleculares (RT-qPCR), além da investigação da população de insetos hematófagos existentes no local. Para pesquisa do VLA e seus anticorpos foi utilizado sangue total e soro dos cervídeos brasileiros provenientes do criatório e dos outros ruminantes domésticos mantidos nas proximidades do criatório. Dos cervídeos que vieram a óbito também foram colhidos fragmentos de órgãos para diagnóstico molecular. Pela técnica de IDGA apresentaram anticorpos anti-vírus da LA: 47,43% (37/78) dos cervídeos, 90,9% (120/132) dos bovinos, 55,49% (96/173) dos ovinos e 19,56% (9/46) dos caprinos. Pelo ELISA competitivo em fase sólida foram reagentes: 38,46% (30/78) dos cervídeos, 93,18% (123/132) dos bovinos, 60,69% (105/173) dos ovinos e 23,91% (11/46) dos caprinos. Pela técnica de RT-qPCR realizada a partir do concentrado de hemácias dos animais foram positivos: 10,14% (7/69) dos cervídeos, 0,75% (1/132) dos bovinos, 10,65% (18/169) dos ovinos e 9,09% (4/44) dos caprinos. Dos 46 cervídeos que vieram a óbito foi realizado também RT-qPCR de órgãos e 8,69% dos animais (4/46) foram positivos para VLA. Entre 2015 e 2016 foram capturados em armadilhas luminosas insetos hematófagos da família Ceratopogonidae, Psychodidae e Simuliidae, sendo negativos para vírus da LA por RT-qPCR. Quanto aos sorotipos circulantes na região, acometendo os animais, sua identificação ainda está sendo conduzida. Conclui-se que o VLA ocorre na região do município de Jaboticabal, uma vez que os animais estudados apresentaram alta soroprevalência e também foi possível detectar o VLA, tanto em ruminantes domésticos e selvagens saudáveis, quanto em cervídeos que vieram a óbito. Foi possível identificar a presença de mosquitos da família Ceratopogonidae, família a qual pertence o Culicoides, que é conhecidamente o vetor da LA. / Bluetongue (BT) is an infectious viral disease that affects domestic and wild ruminants, transmitted by vectors, mosquitoes of the genus Culicoides (Diptera: Ceratopogonidae). The notification is mandatory according to the list of the OIE (World Organisation of Animal Health) and the Ministry of Agriculture, Livestock and Supply of Brazilian Government. Clinical signs and lesions in acute cases of infection are often subtle or nonexistent; and when present are very similar with others hemorrhagic diseases, it is necessary to use complementary techniques for the definitive diagnosis of the disease. This study aimed to perform an epidemiological study of BT disease in captive cervids using serological (solid phase competitive ELISA and double agar gel imunodifusion - AGID) and molecular techniques (RT-qPCR), in addition to the investigation of the population of hematophagous insects in the area. To investigate the Bluetongue virus (BTV) and their antibodies, whole blood and serum from Brazilian captive cervids and domestic ruminants located nearby were used. From cervids that died, fragments of organs were collected for molecular diagnosis. Using the AGID technique performed, the animals presented anti-BTV antibodies: 47.43% (37/78) of cervids, 90.9% (120/132) of cattle, 55.49% (96/173) of sheep and 19.56% (9/46) of goats. By the solid phase competitive ELISA were reagents: 38.46% (30/78) of cervids, 93.18% (123/132) of cattle, 60.69% (105/173) of sheep and 23.91% (11/46) of goats. Using RT-qPCR from the whole blood the positive results were: 10.14% (7/69) of cervids, 0.75% (1/132) of cattle, 10.65% (18/169) of sheep and 9.09% (4/44) of goats. From the 46 deer that died, RT-qPCR was also performed and 8.69% (4/46) animals were positive for BTV. Between 2015 and 2016, bloodsucking insects of the family Ceratopogonidae, Psychodidae and Simuliidae were trapped in luminous traps, being negative for BTV by RT-qPCR. The identification of circulating serotypes in the region that affects these animals is still being conducted. It is concluded that the VLA occurs in the region of Jaboticabal county, since the animals studied showed high seroprevalence and it was also possible to detect the VLA in healthy domestic and wild ruminants and in cervids that died. It was possible to identify the presence of mosquitoes of the Ceratopogonidae family, which belongs to Culicoides, which is known as the vector of BT. / FAPESP: 2015/22851-8 / CNPq: 140626/2013-1

Doctor of Philosophy / Division of Biology / Kristin Michel / The biting midge, Culicoides sonorensis, vectors orbiviruses of economic importance, such as epizootic hemorrhagic disease virus (EHDV). Due to the limitations in available molecular tools, critical Culicoides-orbivirus interactions underlying vector competence remain unclear. To provide a foundation for the study of midge-EHDV interactions, RNA interference (RNAi) was developed as a reverse genetic tool, and EHDV-2 infection dynamics were determined within C. sonorensis. To develop RNAi, exogenous double-stranded RNA (dsRNA) was injected into C. sonorensis adults specific to the C. sonorensis inhibitor of apoptosis protein 1 (CsIAP1) ortholog (dsCsIAP1). A significant decrease in CsIAP1 transcripts was observed in whole midges, with highest reduction in the midgut. In addition, dsCsIAP1-injected midges had increased mortality, a loss of midgut tissue integrity, and increased caspase activity. The longevity and midgut phenotypes were partially reversed by the co-injection of dsRNA specific to the C. sonorensis initiator caspase Dronc ortholog and CsIAP1. These results demonstrated that RNAi can be achieved in the midge midgut through injection of target dsRNAs into the hemolymph. Furthermore, the time course of EHDV-2 infection within C. sonorensis was characterized. EHDV-2 infection was observed in the midgut and secondary tissues, including the salivary glands, by 5 days post-feeding (dpf). These data are consistent with dissemination of EHDV-2 to secondary susceptible tissues throughout the midge via the hemolymph and indicate that virus transmission by C. sonorensis may occur as early as 5 dpf. This work provides a foundation for the future study of Culicoides-orbivirus interactions, including the antiviral role of RNAi at the midgut barrier.

Le travail présenté dans ce manuscrit traite des mécanismes d'émergence des maladies infectieuses. Une première partie du texte est consacrée à la réflexion menée sur ces processus, et à leurs conséquences pour la gestion des maladies. Dans une deuxième partie nous exposons les travaux de modélisation réalisés ; l’objectif est de tester si des changements dans l'expression d'une infection peuvent être expliqués par la dynamique de circulation de l'agent infectieux dans la population hôte, à travers la protection de groupe. Nous montrons que l'apparition de la maladie de Carré chez les lions du Serengeti peut être due à une perte d’immunité de groupe suite à l’extinction du virus. Nous étudions ensuite les conséquences sur le patron épidémique d'une protection partiellement croisée entre souches. Enfin, nous intégrons à cette problématique la structure spatiale de la population hôte, pour expliquer des observations faites sur les systèmes lagomorphes/lagovirus en France. / The study presented here aims to suggest and to test several mechanisms to explain how infectious diseases impact could increase, in relation to microparasite circulation and loss of herd protection. We first introduce the main characteristics of host/microparasite interaction and display some knowledge about emerging infectious diseases, and their consequences for diseases control. Stochastic modelling is then used to study epidemic patterns under several hypotheses and for different host/parasite systems: i) we explain canine distemper emergence in Serengeti lions through a break in virus circulation and the resulting loss of herd immunity; ii) we study the consequences of partial crossprotection induced in hosts by different strains in a small homogeneous host population; and iii) we extend this study to spatially structured host populations to explain data observed in lagomorphs/lagoviruses in France.