Made available in DSpace on 2014-06-11T19:27:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2003-07-04Bitstream added on 2014-06-13T18:31:25Z : No. of bitstreams: 1 luciano_rl_me_jabo.pdf: 288271 bytes, checksum: 77640cc158f0c12048ecdaf245801057 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Um método de diagnóstico baseado na associação entre as técnicas de RT-PCR e ELISA foi desenvolvido para a detecção do vírus da bronquite infecciosa das aves (VBI). Inicialmente, a especificidade e a sensibilidade analíticas dessa técnica, bem como dos métodos de RT-PCR e Nested-RT-PCR, foram determinadas, tendo-se demonstrado, para o primeiro parâmetro, apenas a detecção do VBI, enquanto que outros vírus heterólogos aviários testados, tais como pneumovírus aviário (APV / estirpe do grupo B), vírus da doença de Newcastle (NDV / estirpe La Sota) e vírus da doença de Gumboro (GDV - estirpe Lukert), não foram detectados. Quanto à avaliação da sensibilidade analítica dos métodos empregados neste estudo foi constatado que o RT-PCR-ELISA demonstrou ser 10 vezes mais sensível do que a RT-PCR, enquanto que a reação de Nested-PCR foi 100 vezes mais sensível que a RT-PCR e 10 vezes mais sensível que o RT-PCR-ELISA. A técnica de RT-PCR-ELISA, juntamente com os outros dois métodos de biologia molecular, foram aplicadas para a detecção das estirpes H-120 e A034 do VBI, em amostras de pulmão e traquéia obtidas de aves experimentalmente infectadas com essas duas estirpes virais. Os resultados obtidos nos diferentes métodos de biologia molecular foram comparados entre si e com a técnica padrão de isolamento viral em ovos embrionados, verificando-se que o RT-PCR-ELISA revelou-se tão específico quanto as técnicas de isolamento viral e Nested-PCR, porém foi menos sensível do que esses mesmos métodos na detecção do VBI. A técnica de RT-PCR-ELISA, utilizada pela primeira vez para o VBI, demonstrou o potencial de se constituir uma alternativa viável para um diagnóstico direto mais rápido e específico do VBI. / A molecular biology method for the detection of avian infectious bronchitis virus (IBV) was developed combining the reverse transcription and polymerase chain reaction (RT-PCR) with the enzyme-linked immunosorbent assay (ELISA). Firstly, the analytical specificity and sensitivity of RT-PCR-ELISA, RT-PCR and Nested-RT-PCR were assessed. The specificity of these methods were confirmed since only IBV was detected, while other heterologous avian viral pathogens such as pneumovirus (Group B), Newcastle disease virus (LaSota strain) and gumboro disease virus (Lukert strain) were not detected by these techniques. The sensitivity of these methods was established, indicating that the RT-PCR-ELISA was about ten times more sensitive than conventional RT-PCR, while Nested-PCR was a hundred more sensitive than RT-PCR and ten times more sensitive than PCR-ELISA. The RT-PCR-ELISA and the two molecular biology methods were also applied for the detection of IBV strains H-120 and A034, in lung and tracheal tissue samples collected from experimentally infected chickens. The results of these different methods were also compared with the standard diagnostic technique, the virus isolation in chicken embryonated eggs, showing that RT-PCR-ELISA was as specific as virus isolation and Nested-PCR, however it was less sensitive than these methods for the IBV detection. The RT-PCR-ELISA, applied for the first time for IBV detection and direct diagnosis in tissue samples, can be potentially applied as an useful alternative for the rapid and specific diagnosis of IBV.

Orientador: Hélio José Montassier / Banca: José Moacir Marin / Banca: Liana Brentano / Resumo: Um método de diagnóstico baseado na associação entre as técnicas de RT-PCR e ELISA foi desenvolvido para a detecção do vírus da bronquite infecciosa das aves (VBI). Inicialmente, a especificidade e a sensibilidade analíticas dessa técnica, bem como dos métodos de RT-PCR e Nested-RT-PCR, foram determinadas, tendo-se demonstrado, para o primeiro parâmetro, apenas a detecção do VBI, enquanto que outros vírus heterólogos aviários testados, tais como pneumovírus aviário (APV / estirpe do grupo B), vírus da doença de Newcastle (NDV / estirpe La Sota) e vírus da doença de Gumboro (GDV - estirpe Lukert), não foram detectados. Quanto à avaliação da sensibilidade analítica dos métodos empregados neste estudo foi constatado que o RT-PCR-ELISA demonstrou ser 10 vezes mais sensível do que a RT-PCR, enquanto que a reação de Nested-PCR foi 100 vezes mais sensível que a RT-PCR e 10 vezes mais sensível que o RT-PCR-ELISA. A técnica de RT-PCR-ELISA, juntamente com os outros dois métodos de biologia molecular, foram aplicadas para a detecção das estirpes H-120 e A034 do VBI, em amostras de pulmão e traquéia obtidas de aves experimentalmente infectadas com essas duas estirpes virais. Os resultados obtidos nos diferentes métodos de biologia molecular foram comparados entre si e com a técnica padrão de isolamento viral em ovos embrionados, verificando-se que o RT-PCR-ELISA revelou-se tão específico quanto as técnicas de isolamento viral e Nested-PCR, porém foi menos sensível do que esses mesmos métodos na detecção do VBI. A técnica de RT-PCR-ELISA, utilizada pela primeira vez para o VBI, demonstrou o potencial de se constituir uma alternativa viável para um diagnóstico direto mais rápido e específico do VBI. / Abstract: A molecular biology method for the detection of avian infectious bronchitis virus (IBV) was developed combining the reverse transcription and polymerase chain reaction (RT-PCR) with the enzyme-linked immunosorbent assay (ELISA). Firstly, the analytical specificity and sensitivity of RT-PCR-ELISA, RT-PCR and Nested-RT-PCR were assessed. The specificity of these methods were confirmed since only IBV was detected, while other heterologous avian viral pathogens such as pneumovirus (Group B), Newcastle disease virus (LaSota strain) and gumboro disease virus (Lukert strain) were not detected by these techniques. The sensitivity of these methods was established, indicating that the RT-PCR-ELISA was about ten times more sensitive than conventional RT-PCR, while Nested-PCR was a hundred more sensitive than RT-PCR and ten times more sensitive than PCR-ELISA. The RT-PCR-ELISA and the two molecular biology methods were also applied for the detection of IBV strains H-120 and A034, in lung and tracheal tissue samples collected from experimentally infected chickens. The results of these different methods were also compared with the standard diagnostic technique, the virus isolation in chicken embryonated eggs, showing that RT-PCR-ELISA was as specific as virus isolation and Nested-PCR, however it was less sensitive than these methods for the IBV detection. The RT-PCR-ELISA, applied for the first time for IBV detection and direct diagnosis in tissue samples, can be potentially applied as an useful alternative for the rapid and specific diagnosis of IBV. / Mestre

The first aim of this research was to determine the prevalence of IBV in broilers within the Canterbury province, New Zealand, in late winter and to search for associations with management or environmental factors. The second aim was to study how ambient stressors affect the immune system in birds, their adaptive capacity to respond, and the price that they have to pay in order to return to homeostasis. In a case control study, binary logistic regression analyses were used to seek associations between the presence of IBV in broilers and various risk factors that had been linked in other studies to the presence of different avian pathogens: ambient ammonia, oxygen, carbon dioxide, humidity and litter humidity. Pairs of sheds were selected from ten large broiler farms in Canterbury. One shed (case) from each pair contained poultry that had a production or health alteration that suggested the presence of IBV and the other was a control shed. Overall, IBV was detected by RT-PCR in 50% of the farms. In 2 of the 5 positive farms (but none of the control sheds) where IBV was detected there were accompanying clinical signs that suggested infectious bronchitis (IB). Ambient humidity was the only risk factor that showed an association (inverse) with the prevalence of IBV (p = 0.05; OR = 0.92). It was concluded within the constraints of the totally enclosed management systems described, that humidity had an influence on the presence of IBV, but temperature, ammonia, carbon dioxide, oxygen or litter humidity had no effect. In another study environmental temperatures were changed in order to affect the biological function and adaptive capacity of chickens following infection with IBV. The 'affective states' of the animal were assessed by measuring levels of corticosterone (CORT) in plasma and tonic immobility (TI). It was found that low (10 +/- 2°C) and high (30 +/- 2°C) temperatures exacerbated the respiratory signs and lesions in birds infected with IBV as compared to those housed at moderate (20 +/- 2°C) temperatures. The chickens housed at high temperatures showed significantly decreased growth, a higher proportion of hepatic lesions (principally haemorrhages) and a longer tonic immobility period, but there was no significant alteration in the plasma levels of CORT. The birds housed at low temperatures developed a higher proportion of heart lesions (hydropericardium, ventricular hypertrophy) and had significantly higher levels of plasma CORT than birds housed under moderate and/or high temperatures. The specific antibody response to IBV decreased in birds housed under high temperatures. Interestingly the birds housed at high temperatures developed significantly higher levels of haemagglutinin antibodies to sheep red blood cells (SRBC) than those birds housed under low or moderated temperatures. Cell mediated immunity was not significantly affected by heat or cold stress in the first 13 days of treatment but at 20 days the levels of interferon gamma in the birds subjected to low temperatures were lower than in the high temperature group. In other trials, the exogenous administration of low physiological doses of oral CORT (as compared to high pharmacological doses typically used in such experiments) to birds resulted in suppression or enhancement of the immune response depending on duration of treatment and/or dose and nature of the antigen. To our knowledge, this is the first study to show that exogenous CORT can produce an enhancement in the immune response in chickens. iv In conclusion, environmental stressors such as high or low temperatures do affect the physiology of the fast-growing broiler. The adjustments the birds have to make to maintain homeostasis impacts on the course of common infectious diseases, such as IB, that normally is mild in the New Zealand poultry industry. The administration of exogenous CORT showed that this hormone may be part of the physiological stress response and acts as a messenger to prepare the immune system for potential challenges (e.g., infection).

Orientador: Hélio José Montassier / Banca: Adolorata Aparecida Bianco Carvalho / Banca: Clarice Weins Arns / Banca: Geraldo Aleixo da Silva Passos Júnior / Banca: Liana Brentano / Resumo: As respostas imunes inatas e adquiridas, incluindo-se aí tanto as mediadas por fatores humorais como celulares normalmente induzidas após a infecção ou vacinação com o vírus da BI (VBI), são caracterizadas por sua grande complexidade e por aspectos relevantes que ainda são pouco conhecidos, no que tange aos elementos capazes de exercer uma ou mais ações efetoras contra esse patógeno e que culminassem na restrição da replicação viral, seguido de sua eliminação do organismo hospedeiro e também no impedimento de lesões mais severas. Isso posto, foi formulado o presente estudo com o fito principal de fazer a avaliação das respostas imunes humorais e celulares em diferentes intervalos pós-desafio com o VBI de aves previamente vacinadas ou não, realizando-se a mensuração de anticorpos no soro e na lágrima, e a quantificação da expressão de genes relacionados às respostas imunes na superfície traqueal, a fim de correlacionar tais parâmetros com o estado de proteção ao desafio. Os resultados demonstraram que os aumentos significativos nos níveis de anticorpos lacrimais dos isótipos IgG e IgA nas aves previamente vacinadas e também na expressão dos genes relacionados às respostas imunes, sobretudo o CD8, a Granzima A e o IFNg foram correlacionados negativamente com um ou mais parâmetros de alterações patológicas traqueais. Constatou-se também, que a memória das respostas imunes humorais e cito-mediadas conferida por uma única vacinação contra a BI no primeiro dia de idade é dependente da dose vacinal administrada / Abstract: Avian infectious bronchitis virus (IB) is a worldwide infectious disease which causes significant economic losses in poultry industry. The innate and acquired immune responses, including whether there mediated by both cellular and humoral factors that are induced after infection or vaccination with IB virus (IBV) are characterized by their higher complexity and for the relevant aspects that are still poorly known, with respect to the elements able to exercise one or more actions against the pathogen and that culminate in the restriction of its propagation and also on your clearance of the host organism. So, this project was formulated with the main done to make the evaluation of cellular and humoral immune responses at different intervals post-immunization or postchallenge with IBV poultry previously vaccinated or not, performing the measurement of antibodies in serum or tears and quantitation of the expression of genes related to immune responses in tracheal surface, correlating these parameters with the protection against IBV. The results showed that both significative increase of IgG and IgA isotypes in tears of previously vaccinated chickens and the expression levels of genes related to immune responses, especially CD8, Granzyme A and IFN g were negatively correlated with one or more parameters of pathological lesions at trachea. Moreover, we found that the immune memory conferred by vaccination against BI on the first day of age is dependent on vaccine dose administered / Doutor

Made available in DSpace on 2014-06-11T19:33:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-11-26Bitstream added on 2014-06-13T20:48:10Z : No. of bitstreams: 1 okino_ch_dr_jabo.pdf: 2216742 bytes, checksum: fe4b5f21894b32b436b973278d8ed733 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As respostas imunes inatas e adquiridas, incluindo-se aí tanto as mediadas por fatores humorais como celulares normalmente induzidas após a infecção ou vacinação com o vírus da BI (VBI), são caracterizadas por sua grande complexidade e por aspectos relevantes que ainda são pouco conhecidos, no que tange aos elementos capazes de exercer uma ou mais ações efetoras contra esse patógeno e que culminassem na restrição da replicação viral, seguido de sua eliminação do organismo hospedeiro e também no impedimento de lesões mais severas. Isso posto, foi formulado o presente estudo com o fito principal de fazer a avaliação das respostas imunes humorais e celulares em diferentes intervalos pós-desafio com o VBI de aves previamente vacinadas ou não, realizando-se a mensuração de anticorpos no soro e na lágrima, e a quantificação da expressão de genes relacionados às respostas imunes na superfície traqueal, a fim de correlacionar tais parâmetros com o estado de proteção ao desafio. Os resultados demonstraram que os aumentos significativos nos níveis de anticorpos lacrimais dos isótipos IgG e IgA nas aves previamente vacinadas e também na expressão dos genes relacionados às respostas imunes, sobretudo o CD8, a Granzima A e o IFNg foram correlacionados negativamente com um ou mais parâmetros de alterações patológicas traqueais. Constatou-se também, que a memória das respostas imunes humorais e cito-mediadas conferida por uma única vacinação contra a BI no primeiro dia de idade é dependente da dose vacinal administrada / Avian infectious bronchitis virus (IB) is a worldwide infectious disease which causes significant economic losses in poultry industry. The innate and acquired immune responses, including whether there mediated by both cellular and humoral factors that are induced after infection or vaccination with IB virus (IBV) are characterized by their higher complexity and for the relevant aspects that are still poorly known, with respect to the elements able to exercise one or more actions against the pathogen and that culminate in the restriction of its propagation and also on your clearance of the host organism. So, this project was formulated with the main done to make the evaluation of cellular and humoral immune responses at different intervals post-immunization or postchallenge with IBV poultry previously vaccinated or not, performing the measurement of antibodies in serum or tears and quantitation of the expression of genes related to immune responses in tracheal surface, correlating these parameters with the protection against IBV. The results showed that both significative increase of IgG and IgA isotypes in tears of previously vaccinated chickens and the expression levels of genes related to immune responses, especially CD8, Granzyme A and IFN g were negatively correlated with one or more parameters of pathological lesions at trachea. Moreover, we found that the immune memory conferred by vaccination against BI on the first day of age is dependent on vaccine dose administered

Submitted by Ubirajara Cruz (ubirajara.cruz@gmail.com) on 2017-03-28T13:31:49Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_Giseli_Ritterbusch.pdf: 1007939 bytes, checksum: 6829555e6aeec4b59e73c9a7f0f29087 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-03-28T20:59:38Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_Giseli_Ritterbusch.pdf: 1007939 bytes, checksum: 6829555e6aeec4b59e73c9a7f0f29087 (MD5) / Made available in DSpace on 2017-03-28T20:59:38Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_Giseli_Ritterbusch.pdf: 1007939 bytes, checksum: 6829555e6aeec4b59e73c9a7f0f29087 (MD5) Previous issue date: 2015-01-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / O vírus da bronquite infecciosa das galinhas (VBI) é o agente etiológico da Bronquite Infecciosa (BI), uma enfermidade altamente contagiosa que causa grandes perdas econômicas na avicultura. O VBI é um vírus envelopado, que possui genoma constituído de RNA fita simples, que codifica 4 proteínas estruturais, dentre elas a Nucleoproteína (N), que é produzida em grande quantidade na infecção viral e é reconhecidamente imunogênica. O controle da BI se faz com a imunização das aves através da aplicação de vacinas vivas atenuadas, seguidas de vacinação utilizando antígeno inativado, sendo o sorotipo Massachusetts o único liberado para uso no Brasil. Um dos objetivos do presente trabalho foi realizar um estudo exploratório, afim de conhecer a opinião de diferentes segmentos da avicultura sobre a situação atual da ocorrência de BI nos planteis brasileiros e os custos que ela representa. Diante disso, surge então a necessidade do desenvolvimento de vacinas alternativas e seguras para controle da BI, entre elas a utilização de vacinas vetoriais. Dessa forma, com o objetivo de desenvolver uma vacina efetiva no controle da BI, amostras variantes de VBI foram clonadas em adenovírus humano recombinante e utilizadas para transfectar células HEK293, originando adenovírus recombinantes carreadores do gene N do VBI. Estes vírus foram purificados e utilizados como vacinas recombinantes para imunização de aves SPF. Com base nos dados obtidos, observou-se que apesar das diferentes estratégias de vacinação, a BI ainda é considerada uma doença de alta prevalência que continua causando significativas perdas econômicas na produção avícola de corte e postura no Brasil. Os resultados obtidos demonstraram que a vacina recombinante não induziu uma resposta sorológica detectável pelo teste de Elisa comercial utilizado, bem como não reduziu os escores de lesões nos tecidos das aves vacinadas e desafiadas. Assim, a vacina recombinante carreada por adenovírus defectivo expressando o gene N do VBI foi construída e caracterizada, porém se mostrou ineficaz e não induziu suficiente proteção às aves experimentalmente imunizadas frente ao desafio com VBI. / The infectious bronchitis virus (IBV) is the etiologic agent of Infectious bronchitis (IB), a highly contagious disease that causes great economic losses in the poultry industry. The IBV is an enveloped virus that has RNA single strand genome, encoding four structural proteins, among them Nucleoprotein (N), which is produced abundantly in viral infection and is known immunogenic. The IB control is done by immunization of birds by applying live attenuated vaccine, followed by vaccination using inactivated antigen, wherein the Massachusetts serotype is the only released for use in Brazil. One of the goals of the present work was to conduct an exploratory study in order to know the opinion of different segments of the poultry industry on the current situation of the occurrence of BI in Brazilian squads and the costs that it represents. Therefore, the development of alternative and safe vaccines to BI control is necessary, including the use of vectors. In order to develop an effective vaccine to IB control, samples from IBV field variants were cloned into recombinant human adenovirus and used to transfect HEK293 cells, resulting in recombinant adenovirus carriers of the N gene of the IBV. These recombinant viruses were purified and used as vaccines to immunization of SPF chickens. Based on the obtained data, it was observed that despite the different vaccination strategies, IB is still considered highly prevalent disease that causes significant economic losses in Brazilian poultry industry. The results here obtained showed that the recombinant vaccine does not causes detectable positive serological responses by commercial Elisa test in vaccinated chickens and does not reduce the tissues damage in vaccinated and challenged chickens. Thus, the recombinant vaccine carried by defective adenovirus expressing N gene of IBV was constructed and characterized, but seemed to be ineffective and did not induce sufficient protection to experimentally immunized chickens against IBV challenge.