Este trabalho teve como objetivo geral estabelecer critérios de maior precisão para a interpretação de resultados do diagnóstico sorológico para a Doença de Newcastle realizadas através do teste de HI (inibição da hemaglutinação). A fonte dos dados foram resultados de ensaios sorológicos realizados para monitoria da doença no estado de Santa Catarina, Brasil, no período de 2000 à 2010, sendo estas, cedidas pela CIDASC (Companhia Integrada de Desenvolvimento Agrícola de Santa Catarina). Como metodologia, foram ulitizadas técnicas de geoprocessamento com auxílio do aplicativo Capeiro 7, para estudo de caso, métodos estatísticos para estudo das GMTs (médias geométricas dos títulos) e métodos geoestatísticos, com auxilio do aplicavo ArcGis, com desígnio de avaliar q dependência espacial exercida entre os títulos. Estes métodos permitiram a análise espacial dos títulos sorológicos e correlacionar com os vizinhos mais próximos (unidades de produção integradas de frangos de corte) através de mapeamentos. Diante das informações repassadas para o desenvolvimento da presente pesquisa, no estudo de caso observou-se que os títulos sorológicos não exerciam influência sobre os vizinhos. Em um segundo momento, através das análises estatísticas observou-se um declínio das GMTs ao longo to tempo, podendo ser vizualizado um patamar de estabilização, que se fixou entre 1,14 e 1,60. Por fim, no estudo geoestatístico, pode-se constatar que os títulos sorológicos apresentaram dependência espacial nos dez anos analisados, sendo que, em ensaios de HI realizados com 4 UHA (unidades hemaglutinantes), os títulos 1/2 e 1/4 apresentaram dependência espacial de 12km, quando estudados em raios de 10 km a partir de granjas reprodutoras, as quais recebem vacinação para Doença de Newcastle. Os títulos 1/8 apresentaram dependência espacial de aproximadamente 8 km e os 1/16 de 5 km. Desta forma, foi detectado que, a medida que os títulos aumentavam, a dependência espacial entre eles diminuía, mas ainda sim, exercendo forte influencia sobre os vizinhos. / This study aimed to establish more precise criteria for the interpretation of results of serological diagnosis for Newcastle disease carried through the test of HI (hemagglutination inhibition). The source of the data were performed serologic test results for monitoring of the disease in the state of Santa Catarina, Brazil, from 2000 to 2010, these being provided by CIDASC (Integrated Agricultural Development Company of Santa Catarina). The methodology were used geoprocessing techniques to aid application Capeiro 7 for case study, statistical methods to the study of GMTs (geometric mean titers) and geostatistical methods with the aid of ArcGis software, with design to assess spatial dependence q exercised between titles. These methods allowed the spatial analysis of bonds and serological correlate with the nearest neighbors (production units integrated broiler) via mappings. Given the information passed to the development of this research, the case study showed that the serological not exerted influence on the neighbors. In a second step, through statistical analysis we observed a decline in GMTs over to time and can be visualized a level of stabilization, which was set between 1.14 and 1.60. Finally, the geostatistical study, it can be seen that the serological spatial dependence in ten years, and, in HI assays performed with 4 UHA (hemagglutinating units), securities 1/2 and 1/4 showed dependence space 12km, when studied in radius of 10 km from reproduction farms, which receive vaccination for Newcastle disease. The titres eighth spatial dependence of approximately 8 km and 1/16 of 5 km. Thus, it was detected that, as the titres increased, the spatial dependence between them lessened, but still exerting a strong influence on the neighbors.

Orientadores: Clarice Weis Arns, Fernando Rosado Spilki / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-16T11:29:32Z (GMT). No. of bitstreams: 1 Orsi_MariaAngela_D.pdf: 5800043 bytes, checksum: 7c499fd86a0e94ef1057fc62e1ba024f (MD5) Previous issue date: 2010 / Resumo: O vírus da doença de Newcastle (VDN) é o agente causador de uma das mais importantes doenças em aves e representa uma ameaça para a indústria avícola. O VDN é um membro da família Paramyxoviridae, subfamília Paramyxovirinae, gênero Avulavirus. São vírus envelopados, não segmentados dotados de genoma RNA de fita simples sentido negativo, associado à doença do trato respiratório, digestivo e nervoso das aves. O Controle da DN se baseia em biossegurança, uso de vacinas e detecção precoce de lotes infectados. No presente estudo, examinamos dez vacinas vivas comercializadas no Brasil quanto à sua estabilidade térmica (ET), virulência e imunogenicidade. Em outra etapa do trabalho, investigou-se a soroprevalência perante o VDN em regiões de produção avícola voltada à exportação ou não de produtos aviários, seguido da detecção do VDN em aves migratórias. Os estudos são complementados pela análise filogenética de VDN isolado de aves comerciais, dos resultados alcançados, cumpre ressaltar: i) os testes de ET revelam elevada estabilidade para as vacinas utilizadas no país, mesmo após dois anos de sua fabricação; ii) o grau de proteção conferido por vacinas vivas contra a DN não depende da virulência residual conforme testes de inoculação intracerebral; iii) a soroprevalência contra VDN em aves nas regiões produtoras e exportadoras, foi de 39,1% e foram isoladas 77 amostras virais, sempre com perfil não-patogênico; iv) sorologia realizada em uma segunda oportunidade detectou-se uma soroprevalência de 28,8% e isolamento de 15 amostras virais que também foram caracterizadas como não-patogênicas; v) observou-se numa soropositividade de 41,7 a 84,3% dependendo da região e isolamento de 12 VDN na região Nordeste, caracterizados como não-patogênicas, indicando que nas áreas não exportadoras circulam vírus de baixa patogenicidade; vi) o genoma dos vírus isolados e das vacinas vivas, atesta que os vírus circulantes em aves comerciais são de provável origem vacinal e pertencem à classe II sendo 71,8% do genótipo II ou La Sota-like e 28,2% do genótipo I ou Ulster-like; vii) por último, a caracterização biológica dos isolados de aves migratórias mostram que há circulação de vírus de baixa e alta patogenicidade em nosso território. Portanto, este conjunto de trabalhos evidencia o status do Brasil como país livre da Doença de Newcastle em aves comerciais / Abstract: Newcastle disease virus (NDV) is the agent that causes one of the most important diseases in birds and represents a threat to industrial aviculture. NDV is a member of the Paramyxoviridae family, Paramyxovirinae subfamily, Avulavirus genus. In the present study, live vaccines commercialized in Brazil were examined in regard to their thermostability (TS), virulence and immunogenicity. In another stage of this work, soroprevalence was investigated, with viral isolation and characterization carried out in poultry production regions focused on production for exportation or domestic commercialization, followed by the detection of the virus in migratory birds. The studies were complemented by phylogenic analysis carried out on the isolates from the commercial birds. From the results obtained, it is important to underscore that i) the tests of TS revealed a high stability of the vaccines used in Brazil, even two years after their manufacture; ii) the level of protection given by live vaccines against NDV does not depend on residual virulence, as confirmed by tests of intracerebral inoculation iii) soroprevalence against NDV in regions with production for exportation was 39.1% and it was possible to isolate 77 samples, always with a non-pathogenic profile; iv) on a second opportunity, a soroprevalence of 28.8% was detected, with isolation of 15 samples, also classified as non-pathogenic; v) in the Brazilian Northeast, a seropositivity of 41.7 to 84.3% was observed depend of region, with isolation of 12 NDVs, characterized as non-pathogenic, indicating that virus of low pathogenicity circulates in those areas that do not export; vi) the genome of the isolated virus and vaccine implies that the circulating virus in commercial birds probably originates from the vaccine and belongs to class II, being 71.8% from genotype II or La Sota-like and 28.2% from genotype I or Ulster-like; vii) finally, the biological characterization of isolates from migratory birds showed that there is circulation of virus of low and high pathogenicity in Brazil. However, this set of studies agrees with the status of Brazil as being a country free of Newcastle disease in commercial birds / Doutorado / Ciencias Basicas / Doutor em Clínica Médica

A Doença de Newcastle (DN) é uma enfermidade de etiologia viral e de rápido poder de disseminação. Um grande número de espécies aviárias é susceptível ao Vírus da Doença de Newcastle (VDN). Entre estas aves, o pombo doméstico (Columba livia), tem sido incriminado como hospedeiro e disseminador da DN. Este estudo foi realizado para avaliar o comportamento de pombos frente ao VDN. Foram avaliadas a patogenia da doença e a cinética da RIH de pombos submetidos à vacinação com estirpes vivas (LaSota) e a infecção experimental com estirpe patogênica (São João do Meriti) para galinhas (Gallus gallus), para avaliar os papéis desempenhados por estas como possíveis reservatórios do VDN. A resposta sorológica foi mensurada com a técnica de HI e a eliminação do genoma viral avaliada com a técnica de RT-PCR. Foi observado que as estirpes vacinas produziram títulos elevados de anticorpos, tanto nas aves vacinadas como nas sentinelas. Na infecção experimental, demonstramos que a estirpe patogênica não produziu a doença clínica em pombos, porém promoveu a formação de anticorpos, bem como a eliminação do genoma viral. Também foi comprovada a alta infectividade do agente, tendo em vista que aves sentinelas apresentaram níveis de anticorpos elevados, nos mesmos patamares das aves infectadas. / Newcastle Disease (ND), is a highly contagious disease of viral etiology and several bird species are susceptible this disease. The domestic pigeon (Columba livia), has been regarded as a host and disseminating agent of ND. Therefore, a study was carried out in order to evaluate the responses of pigeons naturally or experimentally infected with this pathogen and the possible role of these birds as potential reservoirs of NDV. The disease pathogenesis and the kinetics of the host humoral immune response were studied in pigeons subjected to vaccination with live NDV strains (LaSota) and to experimental infection with a NDV strain (São João do Meriti) that affects domestic chickens (Gallus gallus). The serological response was measured by HI and the elimination of the viral genome was evaluated by RT-PCR. Vaccine strains induced high antibody levels, both in vaccinated and in sentinel birds. Clinical signs of the disease were not induced by the pathogenic strain in experimentally infected pigeons, although there was antibody production, as well as elimination of the viral genome. The high infectivity of the agent was also confirmed, since the sentinels birds presented high antibody levels, which were similar to the levels produced by infected birds.

Orientadores: Clarice Weis Arns, Luciana Konecny Kohn / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T17:08:20Z (GMT). No. of bitstreams: 1 Sakata_SoniaTatsumi_M.pdf: 14113239 bytes, checksum: 26dfdfbc0c87c2a3a37c6143072df6c6 (MD5) Previous issue date: 2013 / Resumo: Produtos naturais isolados a partir de invertebrados e organismos marinhos tem sido objeto de pesquisas contínuas ao longo dos últimos cinquenta anos, principalmente devido à sua complexidade estrutural e potentes atividades biológicas. O presente trabalho teve como proposta ampliar e aprofundar a investigação de substâncias bioativas em extratos de organismos marinhos, junto ao IQSC-USP/Projeto temático, realizando bioensaios de atividade antiviral no laboratório de virologia da Unicamp. Com o objetivo de realizar uma triagem para pesquisar substâncias com ação antiviral, foram eleitas duas espécies de vírus de destaque na avicultura, o Metapneumovirus aviário (aMPV) e o vírus da Doença de Newcastle (NDV), representantes da família Paramyxoviridae. As propriedades em comum dos vírus dentro das respectivas subfamílias, como a organização genômica, sequências das proteínas e suas atividades biológicas, permitem a utilização do aMPV como modelo de estudo para importantes agentes infecciosos da subfamília Pneumovirinae, e o NDV da subfamília Paramyxovirinae. Para a triagem de compostos antivirais foi realizada a avaliação in vitro na linhagem celular Chicken Embryo Related (CER) para a propagação dos vírus e analisar os resultados de inibição viral frente a diferentes extratos e substâncias. Dos cento e vinte e cinco extratos testados frente ao aMPV, sete demonstraram ser ativos, e seis com alto potencial antiviral. Inicialmente, cento e quarenta e sete extratos foram testados frente ao NDV, porém, o resultado foi inconclusivo devido a problema com o título da amostra viral. Assim, os sete extratos ativos e os seis com alto potencial antiviral contra o aMPV foram testados quanto à capacidade de inibição do NDV. Apesar das similaridades dos vírus da família Paramyxoviridae, os extratos não tiveram atividade contra o NDV, como ocorreu frente ao aMPV. As amostras ativas foram estudadas em três tipos de tratamento a fim de determinar os possíveis mecanismos de ação dos extratos: Pré-tratamento, fases de adsorção e/ou penetração do vírus na célula; Pós-tratamento, etapas intracelulares de replicação do vírus; e Inativação Viral. Pela análise visual do efeito citopático, os sete extratos ativos contra aMPV, quatro interrompem as etapas intracelulares de replicação do vírus, dois agem nas fases de adsorção e/ou penetração do vírus à célula, e um não tinha quantidade suficiente para realizar o teste. Com a finalidade de avaliar os possíveis mecanismos de ação com maior objetividade, menor risco de contaminação e alta especificidade, testou-se a metodologia de reação em cadeia da polimerase (PCR) em tempo real. Utilizando um composto puro (pirocina A) frente ao aMPV em CER, a metodologia se demonstrou eficiente. Esse dado foi confirmando pela diminuição de RNA viral quando ocorre a atividade antiviral, dando indícios de atuação do composto em etapas intracelulares de replicação do aMPV. A detecção de extratos com atividade antiviral nas situações testadas neste trabalho corrobora o valor da biodiversidade marinha como fonte de produtos promissores na terapêutica de enfermidades virais. Portanto, a necessidade de estudos sobre esses vírus e do desenvolvimento de novos insumos a serem utilizados nos seus controles é de grande importância ponderado a crescente projeção da indústria avícola brasileira no comércio mundial / Abstract: Natural products isolated from invertebrates and marine organisms have been studied over the past fifty years, mainly due to their structural complexity and potent biological activities. This project was proposed to broaden and deepen the research of bioactive substances in extracts of marine organisms, with the IQSC-USP group, performing bioassays antiviral activity in the laboratory of virology at Unicamp. In order to perform a screening to search antiviral substances were elected two species prominent in poultry, the avian Metapneumovirus (aMPV) and Newcastle disease virus (NDV), representatives of the family Paramyxoviridae. The common properties of the virus within their respective subfamilies, such as genomic organization, sequences of proteins and their biological activities, allow the use of aMPV as a study model for important infectious agents of Pneumovirinae subfamily, and NDV Paramyxovirinae subfamily. For antiviral screening, compounds were tested in vitro using Chicken Embryo Related (CER) cell lineage for the propagation of the virus and analyze the results of viral inhibition against various substances. Among one hundred twenty five extracts tested against aMPV, seven were actives and six have been shown high antiviral potential against the virus. Initially, one hundred fourty seven extracts were tested against NDV. However, the result was inconclusive due to problems with the titer of the viral sample. Thus, the seven active extracts and six extracts with high antiviral potential against the aMPV were tested for the ability to inhibit NDV. Despite the similarities of viruses of the family Paramyxoviridae, the extracts had no activity against NDV, as occurred against aMPV. The active samples were studied in three types of treatment in order to determine the possible mechanisms of action of the extracts: Pre-treatment, stages of adsorption and / or penetration of the virus into the cell; Post-treatment, intracellular steps of virus replication; and Viral Inactivation. At visual analysis of cytopathic effect, from all 7 extracts active against aMPV, 4 disrupt intracellular steps of virus replication, 2 acts on adsorption and / or penetration stages of the virus into the cell, and 1 was not enough to perform the test. In order to assess the possible mechanisms of action more objectively, lower contamination risk and high specificity, the polymerase chain reaction (PCR) in real time methodology was tested. Using a pure compound (pirocina A) against aMPV in CER cell lineage, the methodology is demonstrated efficient. This was confirmed by the decrease of viral RNA when occurs antiviral activity, an evidence of compound's action on intracellular steps of aMPV replication. The detection of extracts with antiviral activity in this study confirms the value of marine biodiversity as a source of promising products in the viral diseases therapeutic. Therefore, the primordiality for more studies and the development of new inputs to control these viruses is extremely important to increasing of Brazilian poultry industry in world trade / Mestrado / Microbiologia / Mestra em Genética e Biologia Molecular

La maladie de Newcastle (MN) et l'influenza aviaire (IA) sont des maladies virales d'importances majeures. L'aviculture malgache est dominée par de petits élevages familiaux. Plusieurs facteurs de risque d'IA et de MN décrits dans la littérature sont présents à Madagascar. Les objectifs de cette thèse sont de déterminer les facteurs de risque associés à la transmission des virus de la MN et de l'IA ; d'évaluer leur niveau de circulation et leur incidence clinique ; d'étudier le rôle des circuits de commercialisation des volailles dans leur diffusion et de proposer des mesures de surveillance adaptées.Les sites d'étude étaient Antananarivo et son pourtour, et le Lac Alaotra. Une étude exploratoire des facteurs de risque a été effectuée, suivie d'une.surveillance épidémiologique événementielle et d'une épidémiologie participative pour estimer l'incidence clinique des maladies. Le rôle des circuits commerciaux a été évalué par des méthodes d'analyse de réseaux sociaux.La circulation des 2 virus était plus importante au Lac Alaotra, caractérisé par les rizières, les eaux de surface et les palmipèdes. Les élevages de basse-cour et les élevages de poulets à haut niveau relatif de protection sanitaire étaient les plus à risque à la MN Antananarivo. Leurs profils de facteurs de risque étaient l'absence de mesure de biosécurité et un contact avec plusieurs marchés pour le premier ; et une densité élevée, les visites des auxiliaires d'élevage et des collecteurs d'œufs et un contact avec d'autres acteurs pour le second. Il n'y a pas de circulation d'IAH, la MN est la maladie dominante avec un taux de mortalité de 44%. Le rôle du circuit de commercialisation a été confirmé. Les villages ayant les plus fortes connexions commerciales étaient les plus exposés à la MN mais constituaient aussi des super-diffuseurs de virus. L'association de plusieurs méthodes de surveillance a été discutée pour en améliorer la sensibilité, la faisabilité pratique et l'efficacité. / Newcastle disease (ND) and avian influenza (AI) are major poultry viral diseases. Poultry production in Madagascar is dominated by smallholder family system. Several risk factors for both diseases described in the literature are present in Madagascar: The objectives of this thesis were to determine the risk factors associated with transmission of AI and ND viruses; evaluate the intensity of their transmission and their clinical incidence; study the role of poultry trade network in their spread and suggest surveillance adapted to local conditions.Study sites were: Antananarivo and its surrounding and the Lake Alaotra. An exploratory study of risk factors was carried out. Passive surveillance and participatory surveys were combined to assess the incidence and clinical impact of AI and ND. The role of poultry-trade network was assessed using social network analysis methods. Higher circulating level for both viruses was observed in Lake Alaotra which is characterized by large paddy fields and surface water, and a high density of domestic waterfowl. At the farm level, backyard chicken and chicken farms with some biosecurity awareness were associated with higher risk of ND in Antananarivo. Risk factors associated with them were lack of biosecurity measures and contact with many markets for the former, and a high density of poultry, high exposition to the visits of animal health workers, egg collectors and multiple interactions with other poultry stakeholders for the second. There was no evidence of HPAI circulation The major infection was ND with an overall mortality rate of 44%, within observed outbreaks. The role of poultry trade in the spread of these viruses was confirmed. Villages with the highest commercial connections were the most exposed to ND. They were also super-spreaders of these viruses. The relevance of the specific combination of several methods of surveillance was discussed to improve their sensitivity, practical implementation, and effectiveness.

The hemagglutinin-neuraminidase (HN) molecule of Newcastle disease virus (NDV) is an integral membrane glycoprotein that is oriented with its N-terminus in the cytoplasm and its C-terminus external to the infected cell. Single spanning membrane proteins with this type of topology (N-terminus in, C-terminus out) have been classified as Type II glycoproteins, in contrast to the more common Type I glycoproteins, which are oriented in the opposite direction. (C-terminus in, N-terminus out). The membrane integration of HN protein was investigated using a wheat germ translation system to synthesize and integrate HN protein into microsomal membranes in vitro. The insertion and translocation of HN protein into microsomal vesicles was found to occur cotranslationally without signal sequence cleavage. The membrane targeting required both signal recognition particle (SRP) and SRP receptor. Membrane binding assays utilizing HN nascent chain/ribosome/SRP complexes demonstrated that the membrane insertion of HN polypeptide required the presence of GTP, in a way similar to that described for secretory, multispanning and Type I proteins. To investigate further the membrane translocation process of HN protein, the amino terminal region of HN was mutated to determine the role of this region in the membrane integration of HN. The cDNA sequence encoding the bulk of the cytoplasmic tail of the HN glycoprotein was deleted. When transcripts produced from the mutated cDNA were translated in wheat germ extract in the presence of membranes, several abnormalities were identified in the interaction of the mutant protein with membranes. Although translocation and glycosylation of the mutant protein was detected, the efficiency of membrane translocation and the stability of the mutant protein's membrane interaction were reduced. Even though a large proportion of the mutant products remained nontranslocated and unglycosylated, many of these products were inserted into membrane vesicles in a reverse orientation from the wild type HN protein. The aberrant insertion of the mutant protein required both SRP and SRP receptor. Ribosome-bound mutant nascent chains were able to insert into membranes without the addition of GTP or SRP, but this GTP-independent insertion was in reverse. Therefore, the cytoplasmic tail of the HN glycoprotein appears to playa critical role in the maintanence of faithful directionality of the protein's membrane insertion.

Prostate cancer (CaP) is the second leading cause of cancer related deaths in men in the United States. Currently, androgen depletion is an essential strategy for CaP combined with surgery, chemotherapy and radiation. Hormone independent cancer stem cells escaping conventional therapy present a major therapeutic challenge. The available treatment regimens for hormone resistant CaP are only palliative and marginally increase survival. Therefore, novel strategies to eradicate CaP including stem cells are imperative. Oncolytic virus (OV) therapy is a novel approach that overcomes the limitations posed by radiation and chemotherapy. Oncolytic virotherapy of cancer is based on the use of replication competent, tumor selective viruses with limited toxicity. Newcastle Disease Virus (NDV), an avian paramyxovirus, is a safe and promising OV successfully used in many clinical trials. NDV is inherently tumor selective and cytotoxic but replication restricted in normal cells. But, systemically delivered NDV fails to reach solid tumors in therapeutic concentrations and also spreads poorly within the tumors due to barriers including complement, innate immunity and extracellular matrix. Overcoming these hurdles is paramount to realize the exceptional oncolytic efficacy of NDV. Therefore, we engineered the fusion (F) glycoprotein of NDV and generated a recombinant NDV (rNDV) cleavable exclusively by prostate specific antigen (PSA). The rNDV replicated efficiently and specifically only in prostate cancer (CaP) cells but failed to replicate in the absence of PSA. Further, PSA-cleavable rNDV caused specific lysis of androgen independent and dependent/responsive CaP cells with a mean effective concentration (EC50) ranging from 0.01 to 0.1 multiplicity of infection (MOI). PSA retargeted rNDV efficiently lysed three-dimensional prostaspheres, suggesting efficacy in vivo. Also, PSA-cleavable NDV failed to replicate in chicken embryos, indicating absence of pathogenicity to its natural host, chickens. Prostaspheres generated from DU-145 CaP cell line derived xenografts showed self-renewal, proliferative and clonogenic potential in vitro, and exhibited increased tumorigenicity in vivo. Embryonic stem and progenitor cell markers like Nanog, Nestin and CD44 were overexpressed in spheres as compared to the cell line suggesting prostaspheres comprise tumor-initiating cells from CaP. Xenograft and cell line derived prostaspheres were permissive for rNDV replication, when the fusion protein was activated by exogenous PSA. The EC50 against tumor initiating cells was 0.11-0.14 MOI, suggesting an excellent therapeutic margin for in vivo studies. PSA retargeting is likely to enhance the therapeutic index of rNDV owing to tumor restricted replication and enhanced fusogenicity. Our results suggest PSA retargeted rNDV selectively replicates and lyse PSA producing CaP cells including tumor-initiating cells and is a promising candidate for immediate Phase I/II clinical trials. / Ph. D.

La viande de volaille et les oeufs constituent une source de protéines bon marché mais la production avicole est menacée par deux maladies virales, la grippe aviaire hautement pathogène et la maladie de Newcastle, ayant des implications économiques et de santé publique à travers le monde. L'introduction du virus de l'influenza aviaire (AIV) hautement pathogène H5N1 en Afrique en 2006 a souligné la nécessité d'une meilleure compréhension d'AIV en Afrique. Grâce à des études de surveillance, nous avons constaté que le virus H5N1 ne circulait plus après 2008 en Afrique subsaharienne. Toutefois, les analyses phylogénétiques réalisées sur le génome de virus faiblement pathogènes H5N2 trouvés chez des oiseaux sauvages au Nigeria ont révélé des caractéristiques de virus réassortants. La similitude d'un gène avec ceux trouvés dans d'autres virus d'Afrique australe renforce l'idée qu'AIV est capable de persister et circuler en Afrique. Nous avons également montré que de nouvelles souches virulentes du virus de la maladie de Newcastle (NDV) constituent la majorité des souches détectées. Leur distance génétique par rapport aux autres souches de NDV connues, leur diversité génétique et leur dispersion géographique suggèrent que ces souches ont probablement évolué localement, circulent depuis un certain temps dans la région et que le commerce et le mouvement d'animaux ont contribué à leur propagation. Nos résultats suggèrent également que la contribution des oiseaux sauvages à la dispersion des souches virulentes du NDV est probablement limitée. Au Luxembourg cependant, les oiseaux sauvages pourraient être un acteur important pour l'introduction du NDV / Poultry meat and eggs constitute one of the cheap sources of protein around the world but poultry production is threatened by two main viral diseases, highly pathogenic avian influenza and Newcastle disease, with economic and public health implications worldwide. The introduction of highly pathogenic avian influenza H5N1 virus in Africa in 2006 highlighted the necessity of a better understanding of avian influenza virus (AIV) in Africa. Through surveillance studies, we found that H5N1 virus was not circulating anymore in sub-Saharan Africa after 2008. However, phylogenetic analyses performed on the genome of low pathogenic H5N2 viruses found in wild birds in Nigeria revealed that they were reassortants. The similarity of one gene to those found in other AIV viruses from Southern Africa strengthened the hypothesis that AIV may actually persist and circulate in Africa. We have shown that new virulent strains of Newcastle disease virus (NDV) constituted the majority of the strains detected. Their genetic distance compared to other NDV strains, their genetic diversity and their geographic dispersion in West and Central Africa suggested that these strains probably evolved locally, that they circulated for some time in the region and that trade and movement of animals likely contributed to their spread. Our findings also suggested that the contribution of wild birds to the dispersion of virulent strains of NDV was probably limited. In Luxembourg however, wild birds may be an important player for the introduction of NDV strains

A avicultura brasileira é atualmente uma atividade de grande sucesso. A utilização de sistemas de planejamento associados a novas tecnologias, reflete-se no extraordinário crescimento da atividade. A produção brasileira de frango ultrapassou a marca anual de 10 milhões de toneladas, em 2007. O Brasil está entre os três maiores produtores de frango no ranking mundial, junto com Estados Unidos e China. Haja vista a importância que a avicultura representa para o país, pela geração de benefícios sociais e econômicos, o risco que a Doença de Newcastle (DNC) constitui para a avicultura brasileira é enorme. Um surto desta doença em um centro de produção avícola representaria um risco à economia e incidiria de forma negativa nos níveis de consumo de proteína de qualidade e economicamente acessível à população. A fim de estabelecermos um monitoramento do vírus da Doença de Newcastle (NDV) em aves selvagens, livres ou de cativeiro, e aves domésticas não vacinadas, residentes em regiões de elevada confluência migratória aviária no Brasil e em pingüins ao redor da Estação Antártica Comandante Ferraz (EACF), coletamos amostras de swabs orais e cloacais para a posterior análise por PCR em Tempo Real (qPCR), além de sangue para testes sorológicos, tendo como objetivos maiores, contribuir para o fortalecimento dos serviços de defesa sanitária animal, aumentar a capacidade de investigação, e finalmente, atualizar e harmonizar normas e procedimentos para a prevenção e controle da DNC, referenciando-se nas recomendações da Organização Mundial de Sanidade Animal (Office International des Epizooties - OIE). Das 1072 aves amostradas em diferentes regiões do Brasil, 8 (0,75%) apresentaram resultado positivo para o NDV por qPCR, sendo 5 (62,5% das positivas) delas provenientes da região Norte, 2 (25% das positivas) do Nordeste e 1 (12,5% das positivas) da região Sul do Brasil. Na Antártica, dos 100 pingüins estudados, 2 apresentaram resultado positivo para o NDV por qPCR e em cerca de 33,3% dos soros testados foi detectada a presença de anticorpo pelo teste de Inibição da Hemaglutinação (HI). Todas as amostras positivas foram re-analisadas por qPCR específico para cepas mesogênicas e/ou velogênicas, resultando negatividade, corroborando os dados que certificam o Brasil como sendo livre da Doença de Newcastle. / Brazilian chicken meat exports ended 2007 with shipments of 3,3 million tons, which represented a 21% increase in comparison with 2006. These results were the best in the history of the poultry sector in Brazil. The production reached 10.2 million tons, a result that kept the country as the worlds largest chicken meat exporter and the third largest producer, only behind USA and China. Thus the risk of an introduction of the Newcastle disease virus (NDV) into domestic poultry is enormous and will play severe consequences for the economy and poltry industries. In order to provide the surveillance of the NDV in wild, free or captive, and non vaccinated domestic birds from some regions of migratory birds confluence in Brazil and in penguins around the brasilian Antarctic Station Comandante Ferraz, we collected oral and cloacal swabs, for the viral detection by Real Time PCR (qPCR), and blood for the serological test (hemaglutination inhibition test HI). A total of 1072 birds were sampled in diferent regions of Brazil, where 8 (0.75%) shown positive results to NDV, in which 5 (62.5% of positive) were from North region, 2 (25% of positive) were from Northeast and 1 (12,5% of positive) was from South. In the Antarctic 100 penguins were studied, in which 2 were detected the NDV (2% of total). HI test showed that 33.3% of penguins were seropositives for NDV, indicating their previous contact with the pathogen. All the positive samples by qPCR were repeated with primers projected to detect only virulent strains and no sample was positive, indicating the absence of velogenic strains in Brazil. The epidemiological profile was richer with the isolament of positive samples in embrioned chiken eggs specific patogen free and latter nucleotidic genomic sequencing of isolate.

<p>This thesis examines parental condition, how it is traded off against reproduction and how it is displayed in a secondary sexual trait. The studies were performed on nest-box breeding collared flycatchers Ficedula albicollis on the island of Gotland, in the Baltic Sea. Early breeding and high fitness were found to be associated with high levels of glycosylated haemoglobin possibly governed by migratory exertion and infectious disease. In order to test if immune function is expressed in secondary sexual traits and how it is traded off against reproductive effort a series of experiments were performed, in which birds were challenged with an antigen, via a vaccine containing neutralised paramyxovirus. The forehead patch of the male collared flycatcher serves as a badge of status and is under sexual selection. Good condition, as reflected in strong immune response and low levels of blood parasites was found to be associated with bigger patch size. Patch size was also found to vary in size within the same breeding season in a pattern predictable from immune response data. Immune response, in itself, was found to be costly in terms of reduced survival, confirming that trade-offs involving suppression of immune response may increase fitness. Mating effort was found to be traded off against immune function and moult. Experimental brood size manipulations revealed a trade-off females between number of offspring and immune function. Thus I suggest a set of parameters useful for condition estimation. I also show that immune response is costly and, second, that pathogen resistance probably plays an important role in the shaping of secondary sexual traits and life-history decisions.</p>