Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Orientadores: Clarice Weis Arns, Isabela Cristina Simoni / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T00:22:52Z (GMT). No. of bitstreams: 1 Fernandes_MariaJuditeBittencourt_D.pdf: 1711625 bytes, checksum: d2876be51222b1ba7526b13ab7a72795 (MD5) Previous issue date: 2010 / Resumo: A doença infecciosa da bursa (IBD), denominada também doença de Gumboro, é uma doença aguda, imunossupressora, altamente contagiosa de aves jovens e de grande importância econômica para a avicultura. O vírus da doença infecciosa da bursa (IBDV), sorotipo 1, pode ser classificado de acordo com sua antigenicidade e patogenicidade em amostras clássicas virulentas (cv), atenuadas, variantes antigênicas ou muito virulentas (vv). Estas diferenças antigênicas são encontradas na região hipervariável do gene VP2, que é responsável pela indução de anticorpos neutralizantes e também dos possíveis marcadores de virulência que ainda não estão bem estabelecidos. O gene VP1 parece também apresentar um papel na virulência do vírus. Primeiramente, o objetivo do presente trabalho foi a identificação e caracterização molecular de 66 amostras brasileiras de IBDV através da RT-PCR de um fragmento do gene VP2 seguida pela digestão por enzimas de restrição (RE) e posterior confirmação pelo sequenciamento. A análise da RT-PCR/RE classificou 25 isolados como cepas vv e 16 como cepas cv além da classificação de 6 grupo moleculares. O sequenciamento também confirmou esta classificação com a presnça dos aminoácidos (aa) típicos das amostras vv (222A, 242I, 256I e 294I). Em 3 destes amostras vv também se observou mutações únicas que mostram pequenas, mas contínuas alterações dos vvIBDV circulantes nas granjas brasileiras. A arvore filogenética confirmou a origem comum das nossas amostras vv com os isolados de outros países assim como a origem monofilética destas amostras. Posteriormente foi feito a RT-PCR de um fragmento representativo do gene VP1 das amostras positivas para IBDV e a análise das sequências e filogenética. Quatorze amostras vv e três cv tiveram êxito nas sequências analisadas. Treze amostras vv apresentaram as substituições de aa comuns para as amostras vv (145T, 146D, 147N e 242E), exceto um que apresentou a sequência das amostras cv e na filogenia agrupou-se com estas amostras. A árvore a partir da VP1 pressupõe um rearranjo genético deste gene. Esta amostra com perfil do segmento A de amostra vv e do segmento B de cv seria o primeiro relato no Brasil de um rearranjo genético natural. Estes rearranjos de segmentos que também foram observados em amostras de outros países ou que podem ser produzidos em laboratório (quimeras) mostram que o segmento B pode estar contribuindo para a patogênese deste vírus. A origem destes rearranjos pode ser de troca genética com o uso de vacinas vivas ou se aceita a hipótese de que o segmento VP1 dos vvIBDV se originaram de um rearranjo genético de fonte desconhecida, estes rearranjos com segmento vvVP2 e cvVP1, seriam descendentes dos ancentrais dos vvVP1. Apenas um seqüenciamento completo das duas sequências e estudos in vivo poderão caracterizar o papel da VP1 na virulência desta amostra. Assim, o monitoramento contínuo das amostras de IBDV através da caracterização molecular pela análise das sequências dos genes e a detecção de alterações genéticas que possam influenciar a patogenicidade do vírus são de extrema importância, pois geram informações fundamentais que possibilitam e subsidiam o controle desta doença no Brasil / Abstract: Infectious bursal disease virus (IBDV) causes a disease among young chickens of great economic importance to the poultry industry worldwide both for the both mortality as the immunosuppression. Two distinct serotypes, 1 and 2, of IBDV are recognized. Only the serotype 1 is pathogenic for chickens and classified according to the antigenicity and/or pathogenicity in classical virulent (cv) strains, very virulent (vv) strains, antigenic variant strains, and attenuated strains. This classification has been based mainly on the VP2 gene sequence, more specifically on the hypervariable region corresponding to the induction of neutralizing antibodies and the serotype specificity. However, the fundamental molecular basis for pathogenicity is not yet clear. Studies with the VP1 gene have also shown its possible role in this virulence and pathogenicity. Firstly, the aim of the present paper was the molecular characterization of sixty-six Brazilian IBDV isolates from broiler and layers flocks during the period from 1997 to 2005 by RT-PCR followed by restriction enzyme analysis of a fragment from VP2 gene variable region. Sequence and phylogenetic analysis of the positive isolates were also carried out. Twenty-five of the isolates were identified as very virulent (vv) and sixteen as classic virulent (cv). All of vv isolates had the typical amino acid (aa) residues and clustered in a phylogenetic tree with the vvIBDV strains. Three vv isolates presented four common aa substitutions and differed from other vv strains indicating that the vvIBDVs circulating on Brazilian farms are undergoing slight but continuous exchanges. Furthermore, the Brazilian IBDV isolates characterized by the VP2 sequence in cv and vv strains were analyzed by the sequence and phylogeny of the VP1 gene fragment. Our vv isolates maintained clustered with the other vvIBDVs in phylogenetic tree obtained from the VP1 gene and presented the common aa too. The same occurred with the cv isolates. However, one isolate vv showed both characters, cv and vv into VP1 sequence and clustered with the ours and other cv isolates in the tree. This isolate has similar type of a reassortment / Doutorado / Microbiologia / Doutor em Genetica e Biologia Molecular

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

La bursite infectieuse aviaire (IBD) est une des causes majeures de pertes économiques pour l’industrie aviaire. La vaccination est le principal outil de contrôle de cette maladie et les oiseaux susceptibles doivent être vaccinés aussitôt que le niveau des anticorps maternels (MA) anti-IBDV est suffisamment bas. L’estimation du moment de vaccination est habituellement déterminée par la formule de Deventer qui utilise le titre initial de MA anti-IBDV et la demi-vie des anticorps pour prédire l’évolution du titre. Dans la présente étude, l’effet du gain de poids sur la vitesse de disparition des MA a été étudié dans le but de l’utiliser pour prédire la détermination du moment de la vaccination. L’analyse des taux d’anticorps neutralisants par ELISA a montré que les poussins avec une forte croissance avaient un taux de disparition plus rapide des MA que ceux à faible croissance. Une formule pour la prédiction du moment de vaccination contre le IBDV, basée sur le gain de poids et le niveau des MA a été développée et vérifiée. La prédiction du moment de vaccination avec cette formule a montré une haute corrélation avec les titres de MA mesurés par ELISA. Le virus de l’anémie infectieuse aviaire (CIAV) est une cause importante d’immunosuppression chez le poulet augmentant la pathogénicité des infections secondaires et en entraînant une réponse humorale suboptimale et une forte mortalité. D’autre part, l’infections sub-clinique du au CIAV provoque une immunosuppression qui facilite la coinfection par d’autre virus tel que le IBDV. Les effets de la coinfection à J1 avec une souche vaccinale de CIAV CAV-VAC® (Intervet) et à J14 avec une souche faiblement virulente de IBDV isolée au Québec, sur l’état de santé des poussins, sur la persistance virale et sur la réponse immunitaire ont été étudiés autant chez des poussins de 1 jour d’âge exempts d’agents pathogènes specifique (SPF) que ceux provenant d’élevages commerciaux. Les résultats ont montré que l’inoculation de la souche vaccinale du CIAV a entraîné une infection sub-clinique, une persistance virale dans la rate et le thymus, une altération de la thymopoièse et une réponse humorale temporaire chez les poussins SPF. Ces effets ont aussi été mis en évidence chez des poussins d’élevage commerciaux malgré des taux élevés de MA. Lors de l’infection avec la souche de IBDV chez des poussins déjà vaccinés contre le CIAV, la persistance du CIAV dans les organes lymphoïdes a été aggravée par une présence de réponses humorales temporaires contre les deux virus et une altération des populations lymphocytaires dans les organes lymphoïdes. Par contre, la présence des MA contre le CIAV a limité temporairement ces effets. Ces travaux ont mis en évidence des désordres immunitaires cellulaires et humoraux et une persistance virale chez des poussins vaccinés contre le CIAV et co-infectés avec le IBDV. / Infectious bursal disease (IBD) is one of the major causes of economic losses in the chicken industry. Vaccination is the main tool against the disease, and the susceptible birds should be vaccinated as soon as the maternal antibody (MA) becomes low enough to allow the vaccine to break through. Estimation of vaccination time is currently performed by Deventer formula which uses initial anti-IBDV titer and antibody half-life to predict the titer. Considering the increased growth rate of chicken in the last decades and the wide variations of MA, we have examined the effects of chick’s weight gain on MA decline and the use of weight in predicting IBD vaccination time. The virus neutralization test and ELISA results demonstrated that fast-growing birds had a faster rate of antibody decline whereas slow-growing birds demonstrated a slower rate. Based on the effect of weight-gain on maternal antibody decline, a new formula for predicting IBD vaccination time was introduced and tested. The predicted IBD vaccination time made by this weight formula showed higher correlation with the measured ELISA titers in the experiment. Chicken infectious anemia virus (CIAV) is another cause of immunosuppression in chicken which is characterized by increased pathogenicity of secondary infectious agents, sub-optimal antibody responses and mortality. CIAV subclinical infections can result in immunosuppression and enhancement of pathogenicity of co-infecting agents such as infectious bursal disease virus (IBDV). Effects of pathogenic CIAV and IBDV coinfection on chick’s health and immune responses are investigated in different studies. In this study, newly hatched specific pathogen free (SPF) and commercial chicks were vaccinated with CAV-VAC® (Intervet) vaccine and /or inoculated with a low-virulent Québec isolate of IBDV at 14 days post CIAV vaccination. Inoculation of the CIAV vaccinal strain at hatch resulted in subclinical infection associated with viral persistency in spleen and thymus, alteration of thymopoiesis and transient humoral response in SPF chicks. Subclinical infection, viral persistency and lack of antibody responses were also shown in CIAV inoculated commercial chicks with high MA. Infection of the low-virulent IBDV in the CIAV vaccinated SPF chicks lead to extended viral persistence of CIAV in lymphoid organs, transient immune responses to both CIAV and IBDV, and alteration of lymphocytes subpopulation in the lymphoid organs. In the coinfected commercial chicks, presence the CIAV in the lymphoid organs was controlled by MA in the first 1-2 weeks after hatch. Thereafter, the immune disorders, viral persistence and lack of humoral responses almost similar to the coinfected SPF chicks were recorded.

La bursite infectieuse aviaire (IBD) est une des causes majeures de pertes économiques pour l’industrie aviaire. La vaccination est le principal outil de contrôle de cette maladie et les oiseaux susceptibles doivent être vaccinés aussitôt que le niveau des anticorps maternels (MA) anti-IBDV est suffisamment bas. L’estimation du moment de vaccination est habituellement déterminée par la formule de Deventer qui utilise le titre initial de MA anti-IBDV et la demi-vie des anticorps pour prédire l’évolution du titre. Dans la présente étude, l’effet du gain de poids sur la vitesse de disparition des MA a été étudié dans le but de l’utiliser pour prédire la détermination du moment de la vaccination. L’analyse des taux d’anticorps neutralisants par ELISA a montré que les poussins avec une forte croissance avaient un taux de disparition plus rapide des MA que ceux à faible croissance. Une formule pour la prédiction du moment de vaccination contre le IBDV, basée sur le gain de poids et le niveau des MA a été développée et vérifiée. La prédiction du moment de vaccination avec cette formule a montré une haute corrélation avec les titres de MA mesurés par ELISA. Le virus de l’anémie infectieuse aviaire (CIAV) est une cause importante d’immunosuppression chez le poulet augmentant la pathogénicité des infections secondaires et en entraînant une réponse humorale suboptimale et une forte mortalité. D’autre part, l’infections sub-clinique du au CIAV provoque une immunosuppression qui facilite la coinfection par d’autre virus tel que le IBDV. Les effets de la coinfection à J1 avec une souche vaccinale de CIAV CAV-VAC® (Intervet) et à J14 avec une souche faiblement virulente de IBDV isolée au Québec, sur l’état de santé des poussins, sur la persistance virale et sur la réponse immunitaire ont été étudiés autant chez des poussins de 1 jour d’âge exempts d’agents pathogènes specifique (SPF) que ceux provenant d’élevages commerciaux. Les résultats ont montré que l’inoculation de la souche vaccinale du CIAV a entraîné une infection sub-clinique, une persistance virale dans la rate et le thymus, une altération de la thymopoièse et une réponse humorale temporaire chez les poussins SPF. Ces effets ont aussi été mis en évidence chez des poussins d’élevage commerciaux malgré des taux élevés de MA. Lors de l’infection avec la souche de IBDV chez des poussins déjà vaccinés contre le CIAV, la persistance du CIAV dans les organes lymphoïdes a été aggravée par une présence de réponses humorales temporaires contre les deux virus et une altération des populations lymphocytaires dans les organes lymphoïdes. Par contre, la présence des MA contre le CIAV a limité temporairement ces effets. Ces travaux ont mis en évidence des désordres immunitaires cellulaires et humoraux et une persistance virale chez des poussins vaccinés contre le CIAV et co-infectés avec le IBDV. / Infectious bursal disease (IBD) is one of the major causes of economic losses in the chicken industry. Vaccination is the main tool against the disease, and the susceptible birds should be vaccinated as soon as the maternal antibody (MA) becomes low enough to allow the vaccine to break through. Estimation of vaccination time is currently performed by Deventer formula which uses initial anti-IBDV titer and antibody half-life to predict the titer. Considering the increased growth rate of chicken in the last decades and the wide variations of MA, we have examined the effects of chick’s weight gain on MA decline and the use of weight in predicting IBD vaccination time. The virus neutralization test and ELISA results demonstrated that fast-growing birds had a faster rate of antibody decline whereas slow-growing birds demonstrated a slower rate. Based on the effect of weight-gain on maternal antibody decline, a new formula for predicting IBD vaccination time was introduced and tested. The predicted IBD vaccination time made by this weight formula showed higher correlation with the measured ELISA titers in the experiment. Chicken infectious anemia virus (CIAV) is another cause of immunosuppression in chicken which is characterized by increased pathogenicity of secondary infectious agents, sub-optimal antibody responses and mortality. CIAV subclinical infections can result in immunosuppression and enhancement of pathogenicity of co-infecting agents such as infectious bursal disease virus (IBDV). Effects of pathogenic CIAV and IBDV coinfection on chick’s health and immune responses are investigated in different studies. In this study, newly hatched specific pathogen free (SPF) and commercial chicks were vaccinated with CAV-VAC® (Intervet) vaccine and /or inoculated with a low-virulent Québec isolate of IBDV at 14 days post CIAV vaccination. Inoculation of the CIAV vaccinal strain at hatch resulted in subclinical infection associated with viral persistency in spleen and thymus, alteration of thymopoiesis and transient humoral response in SPF chicks. Subclinical infection, viral persistency and lack of antibody responses were also shown in CIAV inoculated commercial chicks with high MA. Infection of the low-virulent IBDV in the CIAV vaccinated SPF chicks lead to extended viral persistence of CIAV in lymphoid organs, transient immune responses to both CIAV and IBDV, and alteration of lymphocytes subpopulation in the lymphoid organs. In the coinfected commercial chicks, presence the CIAV in the lymphoid organs was controlled by MA in the first 1-2 weeks after hatch. Thereafter, the immune disorders, viral persistence and lack of humoral responses almost similar to the coinfected SPF chicks were recorded.