Made available in DSpace on 2015-03-26T13:47:03Z (GMT). No. of bitstreams: 1 texto completo.pdf: 914989 bytes, checksum: 6324bfc7a5398520ac5042a3539b706e (MD5) Previous issue date: 2011-11-22 / Porcine circovirus-2 (PCV2) has been related as the causative agent of the Postweaning Multissystemic Wasting Syndrome (PMWS) and other diseases called porcine circovirus associated diseases (PCVAD). They are responsible for economic losses in pork production worldwide. There is only a few scientific studies describing the infection in other species but swine and their hole at the epidemiologic dynamics of the diseases related to the PCV2. The aim of this study is to investigate the occurrence of infection by the PCV2 in wild mice (Mus musculus and Rattus rattus) captured in hog farms. The capture of the 40 sorted mice was made at 5 pig wean tofinish farms in Minas Gerais, an important state of pork production in Brazil. Samples of tissues (lymph nodes, spleen, kidney, heart and lungs) and blood were collected from the mice. The tissue fragments collected were submitted to immunohistochemistry and Nested PCR. Additionally, samples from spleen and lungs were analyzed by histology assays. Presence of antibodies anti-PCV2 was tested by ELISA assays. Immunohistochemical analysis showed positive prints in 12 animals, mostly on spleen (sub scapular area), lungs (alveolar macrophages) and kidney (inside the tubules). The 12 serum analyzed by ELISA hasn t detected antibodies anti-PCV2. Histopathological analyses revealed in some samples, a multifocal and lympho-neutrophilic interstitial bronchopneumonia, with some node formations. Moreover, spleen samples showed a mild to moderate lymphocyte depletion related to the PCVAD. The Nested PCR assays showed the presence of viral DNA at different tissues from 6 tested rodents. Thus, the results found in this work, indicate that mice from the species Mus musculus and Rattus rattus can be naturally infected by the PCV2 and they would play a hole in the epidemiology of PCVAD. However, more studies are necessary to confirm the transmission of the PCV2 from wild rodents to pigs. / O porcine circovirus-2 (PCV2) é atribuído como um dos agentes relacionados a doenças associadas ao circovírus (PCVAD), ocasionando perdas econômicas significativas na produção mundial de suínos. Poucos trabalhos são realizados a respeito da infecção em outras espécies pelo PCV2 e sua participação na epidemiologia das doenças associadas ao vírus. O propósito desse estudo foi investigar a ocorrência de infecção em roedores peridomésticos das espécies Mus musculus e Rattus rattus pelo PCV2 em granjas comerciais de suínos. Animais dessas espécies foram capturados em importantes centros de produção no estado de Minas Gerais. Amostras de órgãos (linfonodos, baço, rins, fígado, pulmão) e sangue foram coletadas. Os fragmentos de tecidos coletados foram submetidos ao teste de imunohistoquímica e Nested PCR. Adicionalmente, foram realizadas avaliações histológicas em amostras de baço, rim e pulmão. Presença de anticorpos anti-PCV2 foram avaliados pela técnica de ELISA. O teste de imunohistoquímica demonstrou marcações encontradas em 12 animais, principalmente no baço (região subcapsular), no pulmão (macrófagos alveolares) e nos rins (interior dos túbulos). A análise do soro pela técnica de ELISA não detectou anticorpos contra o PCV-2 nas 12 amostras avaliadas.. A histopatologia demonstrou em algumas amostras, uma pneumonia bronco-intersticial neutrofílica e linfocítica, multifocal e moderada, com formação de nódulos linfóides associados a vasos e bronquíolos. No ensaio de nested-PCR foi detectado DNA viral em diferentes tecidos avaliados de seis animais. Os resultados citados demonstram que os roedores domésticos das espécies estudadas podem exercer importante papel na epidemiologia das doenças relacionadas ao PCV2. No entanto, mais estudos são necessários para comprovar a transmissão do PCV2 dos roedores para os suínos.

Made available in DSpace on 2015-03-26T13:47:16Z (GMT). No. of bitstreams: 1 texto completo.pdf: 926741 bytes, checksum: 18d302c22a4907755b01b95cb9c1ea36 (MD5) Previous issue date: 2013-06-04 / Fundação de Amparo a Pesquisa do Estado de Minas Gerais / The diseases associated with PCV2 (PCVAD) require various factors to occur, however, the virus infection is critical to the development of any one of the syndromes. The present study aimed to determine differences in serologic and viremic profiles for PCV2 in the swine breed Piau and a commercial line (Landrace x Large White x Pietrain) at a subclinically infected farm by the virus studied. The experiment was conducted at the Genetic Improvement Pig Farm (GMS), Federal University of Viçosa (UFV), in which it isn´t carried out vaccination against PCV2. This study conducted a cross-sectional sample of sows (> 2 parity), pigs for 1-3 weeks, 3-8 weeks and 8-22 weeks of age. The serum samples were used to measure the level of total antibodies by ELISA and quantitation of viremia by real time PCR. The results showed that, at the age of 3-8 weeks, the Piau breed piglets seroconverted earlier than the commercial line piglets and the Piau breed sows showed lower levels of total antibodies in relation to the commercial line. There were no differences in viremia between the different stages of production within each genetic group or between groups. This work provides evidence that the breed Piau has a different humoral immune response than the commercial line studied when facing a natural PCV2 subclinical infection. The results of this study reinforce the importance of the conservation of native breeds that have not been used for development of high productivity commercial lines. / As doenças associadas ao PCV2 (PCVAD) necessitam de vários fatores para ocorrer, no entanto, a infecção pelo vírus é fundamental para o desenvolvimento de qualquer uma das síndromes. O presente estudo teve como objetivo verificar diferenças nos perfis sorológicos e virêmicos para o PCV2 entre suínos da raça Piau e de uma linhagem comercial (Landrace x Large White x Pietrain) em uma granja subclinicamente infectada pelo vírus estudado. O experimento foi realizado na Granja de Melhoramento Genético (GMS) da Universidade Federal de Viçosa (UFV), na qual não é realizada a vacinação contra o PCV2. O presente estudo realizou uma amostragem transversal em porcas (>2º parto), suínos de 1-3 semanas, 3-8 semanas e 8-22 semanas de idade. As amostras de soro obtidas foram utilizadas para mensuração dos níveis de anticorpos totais por ELISA indireto e quantificação da viremia por PCR em tempo real. Os resultados demonstraram que, na idade de 3-8 semanas, os leitões da raça Piau soroconverteram mais precocemente em relação aos leitões da linhagem comercial e as porcas da raça Piau apresentaram menores níveis de anticorpos totais em relação às da linhagem comercial. Não houve diferença na viremia entre as diferentes fases de produção dentro de cada grupo genético ou entre os grupos. Este trabalho fornece indícios de que a raça Piau apresenta uma resposta imune humoral diferente da desenvolvida pela linhagem comercial estudada diante de uma infecção subclínica natural pelo PCV2. Os resultados obtidos neste estudo reforçam a importância da conservação das raças nativas que não foram utilizadas para formação de linhagens de alta produtividade.

Porcine <i>Circovirus</i> 2 (PCV2) associated diseases (PCVAD) cause economic loss to the global swine industry. Control measures for PCVAD largely depend on the use of PCV2 vaccines. The available commercial PCV2 vaccines contain either inactivated whole virus particles or recombinant PCV2 capsid protein. These preparations most likely contain varying amounts of immune-irrelevant proteins that can cause adverse injection site reactions, with compromised efficacy due to alteration of protective immune epitopes arising during the viral inactivation process. Other constraints include high production cost attributed to propagation of slow growing virus and expression and extraction of recombinant proteins, a requirement for adjuvants, and the induction of a Th2-biased immune response. Hence, development of new PCV2 vaccines is necessary.<p> There are two recommended PCV2 vaccination strategies. They are i. vaccinating sows, which relies on the passive transfer of maternal immunity to offspring, and ii. immunizing young piglets to induce an active immune response. The piglet vaccination has been shown to confer better protection from mortality. Maternal antibody interference to the induction of an active immune response is an obstacle when piglets are vaccinated at an early age. Can we sidestep this maternal antibody interference? To address this issue, I investigated whether a prototypical PCV2 vaccine, parenterally administered, could override maternally-derived PCV2 antibodies in seropositive piglets. The results of this study were not conclusive. However, they laid the foundation for future studies based upon using varying levels of vaccine antigen with different adjuvants, and administered to piglets with defined maternally derived PCV2 antibodies.<p> Subsequently, I examined if a new PCV2 vaccine candidate comprised of bacteriophage lambda particles displaying part of the PCV2 capsid protein could induce anti-PCV2 immunity. Initial experiments showed that pigs do not have pre-existing anti-lambda antibodies and thus will not neutralize display particles used as a vaccine at primary vaccination. I produced and characterized lambda phage particles displaying four immunodominant regions of porcine circovirus 2 (PCV2) capsid protein fused to the lambda capsid protein D i.e., D-CAP, phage display particles. Expression of D-CAP in <i>Escherichia coli</i> (<i>E. coli</i>) and its presence in the vaccine preparation was shown by ELISA and Western blots using anti-PCV2 polyclonal antiserum from a gnotobiotic pig. The vaccine, lambda particles displaying PCV2 capsid protein immunogenic epitopes fused to lambda D protein (LDP-D-CAP), administered without an adjuvant induced both humoral and cellular immunity to PCV2 in conventional pigs, as shown by ELISA, Western blots, virus neutralization assay and delayed type hypersensitivity (DTH) reactions. This work produced the first potential phage vaccine to PCV2. In order to further investigate the feasibility of using the lambda display technology. I produced and characterized two additional lambda display particle preparations, LDP-D-FLAG and LDP-D-GFP, displaying a FLAG tag and the green fluorescent proteins, respectively.

Porcine <i>Circovirus</i> 2 (PCV2) associated diseases (PCVAD) cause economic loss to the global swine industry. Control measures for PCVAD largely depend on the use of PCV2 vaccines. The available commercial PCV2 vaccines contain either inactivated whole virus particles or recombinant PCV2 capsid protein. These preparations most likely contain varying amounts of immune-irrelevant proteins that can cause adverse injection site reactions, with compromised efficacy due to alteration of protective immune epitopes arising during the viral inactivation process. Other constraints include high production cost attributed to propagation of slow growing virus and expression and extraction of recombinant proteins, a requirement for adjuvants, and the induction of a Th2-biased immune response. Hence, development of new PCV2 vaccines is necessary.<p> There are two recommended PCV2 vaccination strategies. They are i. vaccinating sows, which relies on the passive transfer of maternal immunity to offspring, and ii. immunizing young piglets to induce an active immune response. The piglet vaccination has been shown to confer better protection from mortality. Maternal antibody interference to the induction of an active immune response is an obstacle when piglets are vaccinated at an early age. Can we sidestep this maternal antibody interference? To address this issue, I investigated whether a prototypical PCV2 vaccine, parenterally administered, could override maternally-derived PCV2 antibodies in seropositive piglets. The results of this study were not conclusive. However, they laid the foundation for future studies based upon using varying levels of vaccine antigen with different adjuvants, and administered to piglets with defined maternally derived PCV2 antibodies.<p> Subsequently, I examined if a new PCV2 vaccine candidate comprised of bacteriophage lambda particles displaying part of the PCV2 capsid protein could induce anti-PCV2 immunity. Initial experiments showed that pigs do not have pre-existing anti-lambda antibodies and thus will not neutralize display particles used as a vaccine at primary vaccination. I produced and characterized lambda phage particles displaying four immunodominant regions of porcine circovirus 2 (PCV2) capsid protein fused to the lambda capsid protein D i.e., D-CAP, phage display particles. Expression of D-CAP in <i>Escherichia coli</i> (<i>E. coli</i>) and its presence in the vaccine preparation was shown by ELISA and Western blots using anti-PCV2 polyclonal antiserum from a gnotobiotic pig. The vaccine, lambda particles displaying PCV2 capsid protein immunogenic epitopes fused to lambda D protein (LDP-D-CAP), administered without an adjuvant induced both humoral and cellular immunity to PCV2 in conventional pigs, as shown by ELISA, Western blots, virus neutralization assay and delayed type hypersensitivity (DTH) reactions. This work produced the first potential phage vaccine to PCV2. In order to further investigate the feasibility of using the lambda display technology. I produced and characterized two additional lambda display particle preparations, LDP-D-FLAG and LDP-D-GFP, displaying a FLAG tag and the green fluorescent proteins, respectively.

Porcine circovirus associated disease (PCVAD) is currently one of the most economically important diseases facing the global swine industry. Porcine circovirus type 2 (PCV2) is the primary and essential causative agent of PCVAD, but development of clinical disease typically requires co-infection with other swine pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV). The specific mechanisms of co-infection that lead to clinical disease are not fully understood, but immune modulation by the co-infecting viruses is thought to play a critical role. The ability of dendritic cells (DC) infected with PRRSV, PCV2 or both to induce regulatory T cells (Tregs) was evaluated in vitro. DCs infected with PCV2 significantly increased CD4+CD25+FoxP3+ Tregs (p<0.05) and DCs co-infected with PRRSV and PCV2 induced significantly higher numbers of Tregs than with PCV2 alone (p<0.05). This Treg induction was found to be dependent on TGF-β and not IL-10. Further investigation of the in vivo swine immune response to acute co-infection with PCV2 and PRRSV failed to detect activation of Tregs in peripheral blood mononuclear cells (PBMCs) or bronchoalveolar lavage samples. The Treg response to in vitro and in vivo PRRSV challenge in pigs persistently infected with PCV2 or vaccinated against PCV2 was evaluated. There was no significant difference in Tregs in PBMCs among chronically PCV2-infected, vaccinated PCV2 challenged or negative control pigs. However, following in vitro infection of monocyte-derived dendritic cells with PCV2, PRRSV, or both viruses, co-cultured lymphocytes from chronically infected and PCV2 vaccinated pigs had significantly (p<0.05) decreased Treg expression in the virus infected groups compared to the negative controls. In separate experiments, pigs vaccinated against PCV2 and subsequently challenged with an attenuated PRRSV strain and its pathogenic parental strain developed increased CD4+CD25+FoxP3+ Tregs (p<0.05) in PBMC samples compared to uninfected controls, and this correlated with increased suppressor activity and IL-10 expression. The findings from these studies indicate that the interaction of PCV2 and PRRSV in swine modulates the host immune response mediated in part through the activity of Tregs. However, the extent to which Tregs orchestrate a dysregulated immune response in the pathogenesis of PCVAD in vivo remains to be determined. / Ph. D.

Made available in DSpace on 2015-03-26T13:46:46Z (GMT). No. of bitstreams: 1 texto completo.pdf: 618598 bytes, checksum: 78e08690fd4db366e1e37fe332d558bc (MD5) Previous issue date: 2009-02-27 / The porcine circovirus, caused by the porcine circuvirus type 2 (PCV 2), and the porcine enzootic pneumonia, caused by the Mycoplasma hyopneumoniae, are infectious contagious diseases of great importance for worldwide swine production, both causes pneumonias with different presentation patterns. They have cosmopolite distribution and are among the major causes in economic losses. This work was made aiming the evaluation of the co-infection between porcine circovirus 2 (PCV2) and Mycoplasma hyopneumoniae using real time polymerase chain reaction (qPCR), immunohistochemistry and histopathological analyses. 120 lungs fragments, 45 being from fragments with macroscopic lesions (CLM) and 75 being from fragments without macroscopic lesions (SLM) were analyzed by histopathology procedures. Among these, 32 samples were randomly selected (being 16 without macroscopic lesions and 16 with macroscopic lesions) for the immunohistochemistry analyses (for Mycoplasma hyopneumoniae detection) and qPCR (for PCV 2 detection). No significant difference was observed between the groups (SLM and CLM) on the tests, indicating that the absence of macroscopic lesions doesn t reject the possible presence of agents neither the presence of microscopic lesions. The histopatological technique showed itself as an important tool for diagnosis approach. Although the lesion are not patognomonic for the agents in the analyses, they are quite suggestive about the viral and bacterium scene, and these findings, together with the presence of clinical signs and agent detection (more frequently made by qPCR and immunohistochemistry), confirm the diagnosis of each agent isolated or co-infection. Although the samples analyzed by RT-PCR had shown higher number of negative results and low viral quantity, many authors related that lungs are among the organs with the lowest viral quantities of PCV2, being, for that reason, the liver linfonodes, the main target of the PCV2, more indicated for viral quantification. It was verified a positive correlation between the immunohistochemistry analysis and the histopathology analysis using the Pearson statistical test, which confirms the presence of the agent in the histopathology observations. Forward studies using linfonodes samples (target of the PCV2) and samples collected from different lung anatomic regions must be realized for evaluation of a co-infection for more accurate results. / A circovirose suína, causada pelo circovirus suíno tipo 2 (PCV2), e a pneumonia enzoótica suína, causada pelo Mycoplasma hyopneumoniae, são doenças infecto-contagiosas de grande importância na suinocultura mundial, ambas causam pneumonias com diferentes formas de apresentação. Têm distribuição cosmopolita e estão entre as principais causas de perdas econômicas. O presente trabalho foi realizado com o objeto de avaliar a coinfecção entre Circovírus suíno 2 e o Mycoplasma hyopneumoniae, por meio da reação da polimerase em cadeia em tempo real (qPCR), técnica de imunoistoquímica e exames histopatológicos. Cento e vinte fragmentos de pulmões, sendo 45 com lesões macroscópicas (CLM) e 75 sem lesões macroscópicas (SLM) foram analisados pela histopatologia. Destes, foram selecionadas aleatoriamente 32 amostras (sendo 16 sem lesões macroscópicas e 16 com lesões macroscópicas) para a realização dos testes de imunoistoquímica (para a detecção do Mycoplasma hyopneumoniae) e qPCR (para detecção do PCV2). Não foi observada diferença significativa entre os grupos (SLM e CLM) em nenhum dos testes, o que indica que a ausência de lesões macroscópicas não descarta a possibilidade da presença dos agentes nem de lesões microscópicas. A técnica de histopatologia representou uma importante ferramenta para se aproximar ao diagnóstico. Embora as lesões não sejam patognomônicas para os agentes avaliados, elas são bastante sugestivas dos quadros viral e bacteriano, e esses achados, aliados a presença de sinais clínicos e detecção do agente (que são mais frequentemente realizados por qPCR e imunoistoquímica), confirmam o diagnóstico de cada agente isoladamente ou da coinfecção. Apesar das amostras analisadas pelo qPCR terem apresentado grande número de resultados negativos e baixa carga viral, diversos autores relatam que o pulmão é um dos órgãos com menor carga viral de PCV2, sendo desta forma os órgãos linfóides, principais alvos do PCV2, mais indicados para a quantificação viral. Foi verificada uma correlação positiva do teste de imunoistoquímica com o teste histopatológico de acordo com o teste estatístico de Pearson, o que confirma a presença do agente nas lesões observadas na histopatologia. Estudos futuros utilizando a coleta de linfonodos (órgão alvo do PCV2) e amostras coletadas de diferentes regiões anatômicas pulmonares devem ser realizados para avaliar a co-infecção de uma forma mais acurada.