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.

Since 2002, the South African poultry industry has experienced outbreaks of Newcastle disease (ND) caused by a newly introduced virus (NDV) strain belonging to lineage 5d/VIId (“goose paramyxovirus” - GPMV). Control of the disease has proved difficult with commercially available vaccines appearing ineffective. In the first of two studies, broilers chicks were vaccinated with VG-GA vaccine (lineage II), then challenged with both GPMV and a “classic” challenge virus (RCV) of lineage 3d/VIII to compare the efficacy of the vaccine against both strains. In the second study, commercial and SPF hens in lay were vaccinated with La Sota vaccine and challenged with GPMV isolate, and immunohistochemistry staining used to determine the distribution pattern of viral antigen in the oviduct of the hens. The second study also compared the efficacy of cloacal and ocular routes of vaccination. The first study did not detect any statistically significant difference in protection offered by the vaccine against the GPMV strain in comparison to the RCV strain. The protection offered by the vaccine against challenge with both viruses was found to be dosedependant with 106.0 EID50 producing a 100% protection and 94.44% and 13.89% for 104.5 EID50 and 103.0 EID50 vaccination doses respectively. Protected birds did not manifest clinical signs, but still had macropathological lesions in some organs at necropsy. The computed protective doses (PD50 and PD90) for the VG-GA vaccine were 103.51 and 104.38 for GPMV and 103.79 and 104.43 for RCV. Results from the second study showed no clear difference in the protection of the oviduct from challenge with GPMV by either the cloacal and ocular routes of vaccination. Vaccinated birds were fully protected (100%) against challenge by La Sota vaccine, but not against infection and replication of the virus, as birds showed varying degrees of macropathology with numerous stained viral antigens in the oviducts demonstrated by immunohistochemistry. The susceptibility and colonisation of the oviduct of laying hens by both the lentogenic La Sota and the virulent NDV isolates was confirmed, with the uterus being more susceptible than magnum and isthmus. Necrosis and apoptosis of cells of the oviduct were not detected but cellular infiltration, gland dilatation and interstitial oedema were observed. / Dissertation (MSc (Veterinary Science))--University of Pretoria, 2009. / Production Animal Studies / unrestricted

The specific objectives of this study were to determine the Newcastle disease virus (NDV) antibody titres from the chicken sera collected from various districts and provinces of Zambia and to determine the seroprevalence of ND in Zambian backyard chickens. Results showed that 73.9 % of the birds sampled tested positive for Newcastle disease (ND) antibodies. The seroprevalence of Newcastle disease virus (NDV) in Zambian backyard chicken flocks varied among the five provinces sampled, ranging from 82.6 % in Eastern Province to 48.3 % in Luapula Province. The seroprevalence of the virus also varied among the 11 districts sampled, ranging from 91.3 % in Monze District of Southern Province to 22.8 % in Mufulira District of the Copperbelt Province. The results indicated that the seroprevalence of ND in Zambia has increased since the last survey conducted in 1994. The data generated is expected to contribute towards a more clear understanding of the epidemiology of NDV that would ultimately contribute towards an improved ND control programme to benefit all stakeholders in Zambia. An improved ND control programme is expected to enhance flock numbers and ultimately improve the dietary requirements and income needs of many poor households in the country. / Dissertation (MSc)--University of Pretoria, 2012. / Veterinary Tropical Diseases / unrestricted

M.S.

Avian paramyxoviruses (APMVs) consist of twelve distinct serotypes (APMV-1 to -12) isolated from a wide variety of domestic and wild birds. APMV-1/Newcastle disease virus (NDV) is the most characterized and globally important avian pathogen, because of the huge economic loss associated with the disease. However, very little information is known about the pathogenicity of APMV 2-12. APMV expresses six structural and two accessory proteins. The functions of APMV accessory proteins (V and W) are not fully established. Only the function of V protein in NDV is studied so far. V protein was found to be an IFN antgonist and a major virulent determinant of NDV. In this study, we tested for the potential functions of W protein in NDV and fuctions of V protein in other APMV serotypes. Vaccination failure is a major cause for NDV outbreak in developing and tropical countries, because of thermolabile nature of vaccine strains. Thermostable and thermolabile NDV strains exhibit difference in W protein length. In the first part of our study, we mutated the genome of a thermolabile NDV strain to express W protein of different lengths, rescued recombinant viruses by reverse genetics system and tested for thermostability. Our results showed that W protein does not confer thermostability to NDV. In the second part of study, we constructed plasmids expressing APMV -2, -3 and -6V proteins and tested for IFN antagonism by a dual luciferase reporter assay. Our results showed that APMV-3V acts as IFN antagonist by blocking IFN induction and thereby may play an important role in the evasion of innate immunity. / Master of Science

Many oncolytic virotherapies have shown great advantages for rapid, rational design through recombinant DNA technology to facilitate the targeting of a broad spectrum of malignancies. Newcastle disease virus (NDV), an avian paramyxovirus, is naturally tumor-selective and inherently oncolytic. Our approach is to develop NDV-based nanoparticles (VBNP) for oncolytic virotherapy. VBNPs are non-infectious and non-replicating and are relatively safe. We obtained VBNPs by co-expressing matrix (M), hemagglutinin (HN), and fusion (F) proteins of NDV in avian/ mammalian cells. The budding characteristics, size and morphology of VBNPs were similar to authentic virions. As a proof of concept, we engineered the apoptin (VP3) gene of chicken anemia virus in VBNPs and specifically targeted them to folate-receptor bearing tumor cells by surface conjugation to folate. The VBNPs killed tumor cells by apoptosis and induced proinflammatory and chemotactic cytokines. The VBNPs, although not curative, were able to limit the progression of xenotransplanted fibrosarcoma and malignant glioma tumors and provided a survival advantage in nude mice. We also engineered NDV M based particles with nipah virus surface glycorporteins to target ephrin B receptors. NDV based nipah Virus BNPs (NiV-ndBNP) were morphologically similar to authentic NiV virions. NiV glycoproteins were incorporated into the NDV M based particles, despite poor sequence homology in the transmembrane domain and cytoplasmic tails of glycoproteins. Our results suggest that VBNPs could be used to deliver small molecules, tumor antigens, anti-tumor/ reporter genes and also aid in generating tumor specific immunity by rational design. / Master of Science

M.S.

Thesis (M.S.)--Mississippi State University. Department of Chemistry. / Title from title screen. Includes bibliographical references.