La Enfermedad de Newcastle (ENC) es considerada como uno de los principales problemas para la avicultura industrial en el mundo, porque constituye una traba para el comercio internacional de aves y porque puede ocasionar grandes pérdidas económicas. Estas pérdidas pueden ser reducidas drásticamente aplicando correctas medidas de bioseguridad y adecuados programas de vacunación. El presente trabajo tuvo como objetivo evaluar la efectividad de tres programas vacunales contra la ENC en pollos de engorde usando vacunas entéricas. Con tal fin se utilizaron 240 pollos BB de la línea Cobb Vantress 500, distribuidos en 4 grupos experimentales. El grupo 1 fue vacunado al primer día con una viva entérica y una inactivada y, revacunado al día 8 con una vacuna viva entérica. El grupo 2 recibió sólo vacunas entéricas los días 1, 8 y 18. El grupo 3, fue vacunado con una vacuna entérica al primero y octavo día de edad. El grupo 4, no recibió ningún tipo de vacunación. El día 28 todas las aves fueron desafiadas vía ocular con un inóculo conteniendo un virus velogénico de la ENC. Se evaluó mortalidad, signos clínicos, parámetros productivos y respuesta serológica medida por la prueba de ELISA a los días 3, 29 y 45 de edad. Se obtuvo una mejora mayor de 7% en la protección contra el virus de Newcastle (vENC) en los grupos que usaron una vacuna entérica con una inactivada, y en el grupo que usó dos revacunaciones, por sobre el grupo que sólo usó una revacunación. Palabras Clave: Newcastle, virus, vacuna inactivada, vacuna entérica, revacunación / Newcastle Disease (ND) is considered one of the main problems for the poultry industry in the world, because it constitutes an obstacle to international trade in birds and because it can cause great economic losses. These losses can be drastically reduced by applying proper biosecurity measures and appropriate vaccination programs. The present study aims to evaluate the effectiveness of three vaccination programs against ND in chickens using enteric vaccines. For this purpose, 240 Cobb Vantress 500 line baby chickens were distributed in 4 experimental groups. Group 1 was vaccinated on the first day with an inactivated vaccine and days 1 and 8, with an enteric vaccine. Group 2 received only enteric vaccines on days 1, 8 and 18. Group 3, vaccinated with an enteric vaccine the first and eighth day. Group 4, did not receive any vaccination. On day 28 all birds were challenged intraocularly with an inoculum containing a velogenic Newcastle Disease virus (NDV). An evaluation of mortality, clinical signs, production parameters and serological response measured by ELISA on days 3, 29 and 45 of age, was done. There was an improvement of over 7%against NDV in those groups using an enteric vaccine with an inactivated one, and in the group using two revaccinations, over the group that only used one revaccination. Key words: Newcastle, virus, inactivated vaccine, enteric vaccine, revaccination. / Tesis

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Memoria para optar al Título Profesional de Médico Veterinario / En este estudio se evalúa la respuesta inmunitaria de avestruces adultas y en recría, en base al alza de anticuerpos séricos específicos, frente a la inoculación del virus de la Enfermedad de Newcastle (VNC) inactivado, incorporado en adyuvante oleoso (Seppic ®) y en un adyuvante mineral comercial de hidróxido de aluminio (Alhidrogel ®). A su vez, se mide clínicamente la reacción tisular local, a los adyuvantes utilizados. Para lo anterior se desarrollaron inóculos, combinando el antígeno con los adyuvantes, el oleoso y mineral, además de una solución placebo incorporada en idénticos adyuvantes; posteriormente, las aves fueron sometidas a un esquema experimental de 7 semanas, con inoculaciones de los productos desarrollados en las semanas 1, 3 y 5; además de muestreos serológicos todas las semanas, para la medición de anticuerpos específicos contra el VNC mediante la prueba de Inhibición de la Hemoaglutinación (IHA) Como resultado, no se encontró una asociación estadística significativa entre la edad de las aves y los títulos de anticuerpos obtenidos. El uso del adyuvante oleoso generó una respuesta inmune significativamente más alta, que la generada por el adyuvante Alhidrogel. No se encontraron alteraciones dérmicas; tampoco se observó reacciones de hipersensibilidad en las aves en estudio Como conclusión se demostró la efectividad de los productos inoculados, los cuales generaron anticuerpos específicos contra el virus Newcastle. Esta investigación aporta resultados preliminares en el estudio de la inmunización de avestruces frente al VNC en Chile

Thesis (M.S.)--University of Delaware, 2007. / Principal faculty advisor: Jack Gelb, Dept. of Animal & Food Sciences. Includes bibliographical references.

Orientador: Helio José Montassier / Banca: Ricardo Luiz Moro de Sousa / Banca: Antonio Carlos Paulillo / Banca: Ricardo de Albuquerque / Banca: Adolorata Aparecida Bianco Carvalho / Resumo: O vírus da doença de Newcastle (VDN) provoca uma das doenças infecciosas mais importantes para as aves domésticas, devido aos elevados impactos negativos para a saúde aviária e a interposição de barreiras comerciais para a indústria avícola. Isso requer a constante evolução de técnicas cada vez mais eficazes de diagnóstico para esse vírus. Neste contexto, a nucleoproteína (NP) de VDN é um dos componentes antigênicos ideais para uso no imunodiagnóstico, porque é mais conservada e tem uma elevada imunogenicidade, de modo que NP pode melhorar o desempenho de sorodiagnóstico do VDN. Assim, este estudo teve como objetivo determinar os perfis de produção de anticorpos (Acs) anti-VDN dos isótipos IgA, IgM e IgG, usando NP recombinante (NPr) do VDN como um antígeno alvo. Amostras de soro e de lágrima foram colhidas de plantéis comerciais de aves de postura e também de aves SPF infectadas experimentalmente com a estirpe vacinal LaSota do VDN. O método de ELISA indireto, usando a NPr do VDN como antígeno de revestimento, foi padronizado e utilizado de forma bem sucedida para a detecção de Acs de galinha anti-VDN dos isótipos IgG e IgM em amostras de soro, e de ambos os isotipos mais IgA para amostras de secreção lacrimal. Ainda, este método de ELISA com NPr foi capaz de diferenciar amostras positivas das negativas para o VDN de soro e de lágrima, e nas aves submetidas à infecção experimental com a estirpe vacinal LaSota, a soroconversão foi detectada mais precocemente para os anticorpos do isótipo IgA (lágrima) e IgM (lágrima e soro), que alcançaram uma concentração máxima no sétimo dia após a infecção (pi), enquanto que os níveis de anticorpos IgG anti-VDN começaram a ser detectados mais tardiamente e atingiram um pico aos 14 dias pi. Além disso, a aplicação do ELISA em amostras de soro e de lágrima colhidas a partir de plantéis comerciais de galinhas de postura, para detecção de dois... / Abstract: Newcastle disease virus (NDV) causes one of the most important infectious diseases for chickens, due to the high negative impacts for health and the demands of trade barriers to poultry industry. This requires the constant development of increasingly more effective diagnostic techniques. In this context, the nucleoprotein (NP) of VDN is one of the ideal antigenic components for immunodiagnostics, because it is more conserved and has a high immunogenicity, so that NP may improve the performance of serodiagnosis of NDV. Thus, this study aimed to determine the production profiles of anti-NDV antibodies of IgA, IgM and IgG isotypes, using recombinant NP (rNP) of VDN as a target antigen. Serum and tear samples were collected from commercial poultry flocks, and from SPF birds experimentally infected with the LaSota vaccine strain of NDV. The indirect ELISA method using the NPr VDN as coating antigen was standardized and used for the detection of anti-NDV chicken antibody isotypes IgG, IgM in serum samples and antibodies of both isotypes added of IgA isotype for lacrimal secretion samples. This ELISA method with NPr was effectively able to differentiate NDV-positive and NDV-negative serum and tear samples, and in the birds subjected to a experimental infection with vaccine strain, the seroconversion was detected earlier for the antibodies of the IgA (tear) and IgM (tear and serum) isotypes, which have reached maximum concentration on the 7th day post-infection (pi), while the IgG anti-NDV antibody levels began to be detected later and peaked at 14 days pi. Moreover, the application of ELISA in serum and lachrymal samples collected from commercial layer chickens,with the detection of two (IgG and IgM for serum samples) or three isotypes (IgG, IgM and IgA for lachrymal samples) of anti-NDV antibodies, resulted in the highest detection frequencies than each antibody isotype was individually detected. By comparing the results of serum samples for ... / Doutor

Made available in DSpace on 2015-10-06T13:03:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-06-15. Added 1 bitstream(s) on 2015-10-06T13:18:23Z : No. of bitstreams: 1 000848339.pdf: 757255 bytes, checksum: 9dcc3acba66df8e1de6d584184ea3dc8 (MD5) / O vírus da doença de Newcastle (VDN) provoca uma das doenças infecciosas mais importantes para as aves domésticas, devido aos elevados impactos negativos para a saúde aviária e a interposição de barreiras comerciais para a indústria avícola. Isso requer a constante evolução de técnicas cada vez mais eficazes de diagnóstico para esse vírus. Neste contexto, a nucleoproteína (NP) de VDN é um dos componentes antigênicos ideais para uso no imunodiagnóstico, porque é mais conservada e tem uma elevada imunogenicidade, de modo que NP pode melhorar o desempenho de sorodiagnóstico do VDN. Assim, este estudo teve como objetivo determinar os perfis de produção de anticorpos (Acs) anti-VDN dos isótipos IgA, IgM e IgG, usando NP recombinante (NPr) do VDN como um antígeno alvo. Amostras de soro e de lágrima foram colhidas de plantéis comerciais de aves de postura e também de aves SPF infectadas experimentalmente com a estirpe vacinal LaSota do VDN. O método de ELISA indireto, usando a NPr do VDN como antígeno de revestimento, foi padronizado e utilizado de forma bem sucedida para a detecção de Acs de galinha anti-VDN dos isótipos IgG e IgM em amostras de soro, e de ambos os isotipos mais IgA para amostras de secreção lacrimal. Ainda, este método de ELISA com NPr foi capaz de diferenciar amostras positivas das negativas para o VDN de soro e de lágrima, e nas aves submetidas à infecção experimental com a estirpe vacinal LaSota, a soroconversão foi detectada mais precocemente para os anticorpos do isótipo IgA (lágrima) e IgM (lágrima e soro), que alcançaram uma concentração máxima no sétimo dia após a infecção (pi), enquanto que os níveis de anticorpos IgG anti-VDN começaram a ser detectados mais tardiamente e atingiram um pico aos 14 dias pi. Além disso, a aplicação do ELISA em amostras de soro e de lágrima colhidas a partir de plantéis comerciais de galinhas de postura, para detecção de dois... / Newcastle disease virus (NDV) causes one of the most important infectious diseases for chickens, due to the high negative impacts for health and the demands of trade barriers to poultry industry. This requires the constant development of increasingly more effective diagnostic techniques. In this context, the nucleoprotein (NP) of VDN is one of the ideal antigenic components for immunodiagnostics, because it is more conserved and has a high immunogenicity, so that NP may improve the performance of serodiagnosis of NDV. Thus, this study aimed to determine the production profiles of anti-NDV antibodies of IgA, IgM and IgG isotypes, using recombinant NP (rNP) of VDN as a target antigen. Serum and tear samples were collected from commercial poultry flocks, and from SPF birds experimentally infected with the LaSota vaccine strain of NDV. The indirect ELISA method using the NPr VDN as coating antigen was standardized and used for the detection of anti-NDV chicken antibody isotypes IgG, IgM in serum samples and antibodies of both isotypes added of IgA isotype for lacrimal secretion samples. This ELISA method with NPr was effectively able to differentiate NDV-positive and NDV-negative serum and tear samples, and in the birds subjected to a experimental infection with vaccine strain, the seroconversion was detected earlier for the antibodies of the IgA (tear) and IgM (tear and serum) isotypes, which have reached maximum concentration on the 7th day post-infection (pi), while the IgG anti-NDV antibody levels began to be detected later and peaked at 14 days pi. Moreover, the application of ELISA in serum and lachrymal samples collected from commercial layer chickens,with the detection of two (IgG and IgM for serum samples) or three isotypes (IgG, IgM and IgA for lachrymal samples) of anti-NDV antibodies, resulted in the highest detection frequencies than each antibody isotype was individually detected. By comparing the results of serum samples for ...

Made available in DSpace on 2014-06-11T19:30:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-02-27Bitstream added on 2014-06-13T21:00:44Z : No. of bitstreams: 1 oliveira_es_me_jabo.pdf: 649478 bytes, checksum: d2bee0b53ef4e30c3486df035f84bee1 (MD5) / Existem diversos trabalhos publicados sobre métodos de diagnóstico da Doença de Newcastle (DN) em aves comerciais da espécie Gallus gallus. Entretanto, é escassa a literatura sobre o desenvolvimento de técnicas para o diagnóstico laboratorial da infecção pelo vírus da Doença de Newcastle (VDN) em espécies de aves não-galiformes. O presente trabalho foi delineado com o objetivo de desenvolver e aplicar a técnica de bloqueio de fase líquida de ELISA com a concanavalina A (BFL-Con A-ELISA) para a detecção e quantificação de anticorpos contra o VDN em soros de pombos de vida livre e da técnica de Semi-nested-RTPCR para detecção do vírus da DN (VDN) em suabes cloacais das mesmas aves. Na comparação entre o BFL-Con A-ELISA e o HI para a detecção de anticorpos em 107 amostras de soros de pombos, foram obtidos uma correlação significativa com o teste de HI (r = 0,875), bem como valores elevados de sensibilidade (100%), especificidade (95,8%), acurácia (96,3%) e concordância (κ = 0,83). Os resultados obtidos na técnica de semi-nested-RT-PCR mostraram que nove das 101 amostras analisadas foram positivas para o VDN. Na análise comparativa dos resultados da técnica do semi-nested-RT-PCR com os obtidos nas técnicas sorológicas, foram observados índices de especificidade relativa de 93,9% e 96,8% em relação aos métodos de HI e BFL-Con A-ELISA, respectivamente; além de uma máxima sensibilidade relativa (100%), comparativamente a esses testes. Esses dados demonstram que tanto o BFL-Con A-ELISA como o semi-nested-RT-PCR desenvolvidos no presente estudo foram eficientes no diagnóstico da DN, ou detectando anticorpos anti-VDN, ou esse agente viral, respectivamente, e ambas as técnicas apresentaram um grande potencial para serem usadas com vantagens no diagnóstico da infecção pelo VDN em pombos e em outras espécies de aves não-galiformes. / A number of studies were done on diagnostic methods of Newcastle disease in commercial poultry (Gallus gallus, species). However the literature is scarce on the development of techniques for the diagnosis of Newcastle disease (ND) infection in non-galiforme and free-living birds. The present study aims to develop and apply a liquid-phase blocking ELISA with Concanavalin A (BFL-Con AELISA) for the detection and quantification of antibodies against ND virus (NDV) in sera of free-living pigeons and a semi-nested-RT-PCR technique for detection of NDV in cloacal swabs collected from these birds. The results of BFL-Con A-ELISA and HI were compared for the detection of antibodies in serum samples from 107 pigeons, and a significant correlation with the HI test was obtained (r = 0.875) as well as high values of relative sensitivity (100 %), specificity (95.8%), accuracy (96.3%) and agreement (k = 0.83). The results of semi-nested-RT-PCR showed that nine of the 101 samples were positive for the presence of NDV. By comparing the seminested- RT-PCR with serological tests, high relative specificity values of 93.9% and 96.8% were obtained with regard to HI and BLF-Con A-ELISA tests, respectively, as well as a maximum relative sensitivity (100%) was observed regarding these serological tests. These data demonstrate that the BFL-Con A-ELISA and seminested- RT-PCR developed in this study were effective in the diagnosis of NDV infection, detecting either specific antibodies, or this virus, respectively, and have a great potential to be used advantageously in the diagnostic of NDV infection in pigeons and other species of non-galliforme birds.

La maladie de Newcastle est une de deux pestes aviaires qui, comme l’influenza, impactent fortement les élevages d’oiseaux domestiques par leur incidence clinique et leurs conséquences économiques sur la filière (contrôle des mouvements d’animaux, abattages sanitaires et préventifs). Des vaccins contre cette maladie ont été développés il y a plusieurs décennies à base de souches virales isolées dans les années 60. Ils assurent normalement une excellente protection clinique. Toutefois, depuis une dizaine d’années, des observations de terrain, principalement en Afrique et en Asie, font état d’échecs partiels de vaccination avec occurrence de foyers réduits dans des élevages a priori correctement vaccinés. En parallèle, des essais in vivo en conditions contrôlées ont établi que les vaccins actuels protégeaient bien cliniquement contre une épreuve avec des souches virulentes récentes mais n’empêchaient pas leur excrétion par les animaux vaccinés. Pour résoudre cette problématique, l’objectif de ce travail a été de générer une souche vaccinale plus efficace contre les souches virulentes circulant actuellement à l’échelle du globe. Pour générer des virus atténués modifiés, nous avons dû dans un premier temps améliorer le système conventionnel de génétique inverse. Nous montrons que la réduction du nombre de plasmides à 2 dans le système, permet de générer plus de virus atténués que le système conventionnel basé sur 4 plasmides. Dans un second temps, nous nous sommes intéressés à étudier le comportement in vitro de virus atténués et virulents équipés de marqueurs fluorescents. Nous montrons que seuls les virus virulents induisent un effet cytopathique in vitro. En revanche, les deux types de virus induisent une infection persistante à long terme sans effet cytopathique. Les cellules infectées de façon persistante résistent à une surinfection par un autre virus. En revanche, lors de co-infections simultanées, nous établissons qu’une cellule infectée par un premier virus peut s’infecter par un second virus lors d’un transfert direct de matériel viral d’une cellule à une autre par des extensions membranaires. Cette observation est remise en perspective par rapport à la capacité de ces virus à se recombiner chez l’animal telle qu’identifiée par des analyses bioinformatiques comparatives de différents isolats. En effet, nous montrons que des cellules peuvent s’infecter avec plusieurs virus par contact direct. Dans un dernier travail, une nouvelle souche vaccinale a été générée consistant à insérer des antigènes immunoprotecteurs d’un virus original isolé à Madagascar en 2008, dans un génome d’une souche vaccinale conventionnelle utilisée depuis plus de 50 ans. Nous montrons que cette nouvelle souche protège efficacement contre trois génotypes viraux dont deux circulant actuellement en Afrique et en Asie. / In addition to influenza, Newcastle disease is one of the two major diseases of poultry that strongly impact the animal health and farming owing to animal bans and depopulations. Vaccines against Newcastle disease are available. They have been developed some decades ago from isolates collected in the 60’s. They usually provide an excellent clinical protection. However, field reports of the last decade, mainly from Africa and Asia, suggest partial vaccination failures in some farms despite proper vaccination. In parallel, in vivo trials have shown that current vaccines provide a good clinical protection against a challenge with recent field strains, but do not prevent shedding of the challenge virus from vaccinated birds. To address this issue, one of the objectives of this study was to generate a new vaccine prototype with improved efficacy against virulent strains circulating worldwide. To generate new engineered attenuated viruses, we first developed an improved reverse genetics system. We demonstrate that the reduction of the number of plasmids to 2 compared to the conventional system based on 4 plasmids does not affect the performances of reverse genetics for virulent strains but significantly increases the yield of attenuated viruses. In a second step, we focused on the behavior of the attenuated and virulent viruses generated by reverse genetics. The viruses were tagged with fluorescent reporter genes to make easier they follow up in cell culture. We show that only virulent strains produce cytopathic effects in vitro. However, both attenuated and virulent strains are able to establish persistent infection in cells without cytopathic effects. Persistently infected cells resist to a super-infection by another virus. In contrast, after co-infection by two different viruses, we show that a cell infected by one virus can be infected by a second one by direct virus trafficking between the cells through cell membrane extensions. This observation supports the possibility of recombination events in the field which are frequently claim in the literature from comparative bioinformatics of field isolates and vaccine strains. Indeed, we show that cells can be infected by multiple viruses through direct contacts between cells. In a last step, a new vaccine prototype has been produced consisting in the substitution of immune-protective antigens in the conventional LaSota vaccine by their homologues derived from an original isolate of Madagascar (2008). We show that this prototype is protective against challenges with three different viruses, including two recent isolates from Africa and Asia.

M.S.