As doenças respiratórias constituem um sério problema em sistemas intensivos de criação de suínos, causando enormes prejuízos à industria suína no Brasil e no mundo. Estes prejuízos estão freqüentemente relacionados à redução de peso, mortalidade, maior predisposição a doenças entéricas, gastos com vacinas e medicamentos. Os distúrbios respiratórios em suínos são manifestados através de um complexo de doenças, com envolvimento de agentes virais, bacterianos e fúngicos. Dentre estes, a presença do circovírus (PCV2) e o Pneumocystis sp. começa a ser gradativamente caracterizada como uma associação entre um agente causador de imunossupressão e um organismo de ação oportunista. O trabalho objetivou diagnosticar Pneumocystis sp. através das técnicas de imunohistoquímica, Grocott e nested-PCR, em suínos abatidos nos Estados do Rio Grande do Sul (RS) e Mato Grosso (MT), diagnosticar a ocorrência de PCV2 na mesma população de suínos, verificar a associação entre Pneumocystis sp. e PCV2, e determinar as relações filogenéticas entre as amostras de Pneumocystis sp. O estudo avaliou um total de 591 pulmões, 297 com alterações macroscópicas (pneumonia) e 294 normais obtidos em frigoríficos. Foram analisados 292 pulmões procedentes do RS e 299 pulmões do MT Para diagnóstico do Pneumocystis sp. as amostras foram analisadas através das técnicas de Grocott, Imunohistoquímica e nested-PCR (mtLSU e mtSSU rRNA). Do total das amostras, 36,9% foram positivas para Pneumocystis. O índice de positividade para o vírus PCV2 foi de 32,7% na amostra total. Os resultados revelaram uma alta prevalência do vírus (PCV2) em pulmões sem lesões macroscópicas. A co-infecção (PCV2 e Pneumocystis sp.), foi detectada em 28,0% em 564 pulmões examinados. As análises das seqüências dos nucleotídeos dos produtos de PCR dos genes mtLSU e mtSSU do rRNA do Pneumocystis sp. nos pulmões analisados, sugerem a presença até o presente momento de 2 espécies diferentes de Pneumocystis no Brasil. Este estudo evidencia a ocorrência da co-infecção de dois agentes (Pneumocystis sp. e PCV2) em animais hígidos, fato que, indica a necessidade de planejamento e implementação de medidas de controle para melhorar a produtividade na suinocultura. / Respiratory diseases are a major problem in intensive systems of swine husbandry. They are a cause for high losses in the swine industry in Brazil and in the world. These losses are often related to weight reduction, mortality, higher vulnerability to enteric diseases, and expenses with vaccines and drugs. Respiratory diseases in swine appear through a complex of diseases, caused by virus, bacteria and fungi; among these, the porcine circovirus 2 (PCV2) and Pneumocystis sp., the former an agent which causes immunosupression and the latter an oportunistic microorganism. Both are being recognized as capable of being associated. The objectives of this study were to: identify Pneumocystis sp. through immunohystochemistry techniques, Grocott and nested-PCR in swine slaughtered in the States of Rio Grande do Sul and Mato Grosso (MT); investigate PCV2 in the same swine population; investigate the association between Pneumocystis sp. and PCV2, and establish a filogenetic relationship between isolated of Pneumocystis sp. The study was carried out with a total of 591 lungs, 297 with macroscopic alterations characteristic of pneumonia, and 294 normal lungs from the industry. 292 lungs came from RS and 299 lungs came from MT. In order, to diagnose Pneumocystis infection, samples were analysed through Grocott technique, immunohystochemistry and nested-PCR (mtLSU and mtSSU rRNA). Among all samples 36,9% were positive for Pneumocystis sp.. PCV2 virus was found in 37,2% of the samples. Results revealed a high prevalence of the PCV2 virus in lungs without macroscopic lesions. Co-infection (PCV2 and Pneumocystis sp.) was found in 28,0% of 564 lungs examined. So far, the analyses of the sequences of nucleotides from the products of PCR from the genes mtlSU rRNA and mtSSU rRNA from Pneumocystis obtained from the examined lungs suggest that, it is possible the existence of two different species of Pneumocystis in Brazil. This study shows co-infection by two agents (Pneumocysts sp. and PCV2) in apparently healthy animals. This fact points out the necessity planning and implementation of control measures in order to improve productiviy in swine husbandry and industry.

As doenças associadas ao circovírus suíno (DACS) causam impacto econômico negativo nos sistemas de criação de suínos no mundo todo. As perdas incluem tratamento da doença, mortalidade, bem como diminuição no desempenho dos animais. Uma das manifestações mais relevantes das DACS é a síndrome multissistêmica do definhamento dos suínos (SMDS). O principal agente patogênico na causa da SMDS é o circovírus suíno tipo 2 (PCV2), no entanto, estudos observacionais e experimentais demonstraram que outros agentes estão envolvidos na patogênese e manifestação dos sinais clínicos. O sequenciamento de alto desempenho aliado a metagenômica são ferramentas que tornam possível a identificação da microbiota total de uma determinada amostra, independentemente de cultivo dos microrganismos. Visando contribuir para o conhecimento dos possíveis vírus envolvidos na SMDS, o presente trabalho realizou o sequenciamento genético de alto desempenho de soros de suínos e posterior análise do metagenoma resultante. Foram utilizadas amostras de soro coletadas em 2008, de 16 suínos com sinais clínicos da SMDS, entre 80 e 100 dias de idade, em uma granja no interior do Rio Grande do Sul. Os dados revelaram sequências virais de PCV2, parvovírus suíno tipo 1 a 6 (PPV1-6), torque teno vírus de suínos (TTSuV) tipo 1b, k2a e k2b e vírus de DNA circular de fita simples codificador de proteína associada a replicação (CRESS). A ocorrência de PCV2, PPV1-5 e TTSuV já foi descrita em suínos com SMDS, portanto este estudo reforça resultados anteriores. O PPV6 foi recentemente descrito na China, Europa e Estados Unidos, e os estudos não relacionaram o vírus com nenhuma doença específica de suínos. Os CRESS já foram identificados em todos os continentes, em vários tipos de amostras, incluindo fezes de suínos, mas sem nenhuma conexão com doenças de animais. Portanto, este é o primeiro relato de PPV6 e CRESS em suínos apresentando sinais de SMDS. Porém estudos posteriores são necessários para poder atribuir relação entre PPV6 e CRESS no desenvolvimento da SMDS. / Porcine circovirus associated disease (PCVAD) is one of the causes of negative economic impact on pig farming systems described worldwide. Losses include expenditures with treatment, increased mortality rates, and decreased productivity. One of the most relevant manifestations of PCVAD is the post-weaning multisystemic wasting syndrome (PMWS). The main pathogen present in PMWS is porcine circovirus type 2 (PCV2). However, observational and experimental studies have shown that other agents may be involved in the pathogenesis and clinical manifestation. High-throughput sequencing combined with metagenomics analyses make it possible to identify the total microbiota in a given sample, regardless of microorganism culture. In order to contribute to the knowledge of the viruses involved in PMWS, the present study carried out the high-throughput sequencing of swine sera and subsequent analysis of the resulting metagenome. Sixteen serum samples collected in 2008 on a farm in Rio Grande do Sul, from 80 and 100 days old pigs with clinical signs of PMWS, were examined. Data revealed viral sequences of PCV2, porcine parvovirus type 1 through 6 (PPV1-6), torque teno sus virus (TTSuV) types 1b, k2a and k2b and circular replication-associated protein (Rep) encoding single-stranded (CRESS) DNA viruses. The occurrences of PCV2, PPV1-5 and TTSuV have already been described in pigs with PMWS, so this study reinforces previous results. PPV6 was recently described in China, Europe and the United States, and the studies did not correlate the virus to any specific disease. CRESS DNA viruses have been identified on all continents in various types of samples, including swine feces, but without any connection to animal diseases. The present study is the first report of PPV6 and CRESS in pigs presenting PMWS signals. However, further studies are necessary to be able to attribute the relationship between PPV6 and CRESS in the development of SMDS.

A Síndrome Multissistêmica do Definhamento do Suíno (SMDS) é uma doença emergente e mundialmente distribuída, que tem trazido sérios prejuízos econômicos para a indústria suinícola. O Circovírus Suíno tipo 2 (PCV2), agente causal da doença, provoca lesões principalmente nos tecidos linfóides, e sugere-se que produza imunossupressão, predispondo o hospedeiro a infecções virais, bacterianas e fúngicas secundárias. Neste trabalho, é descrito um estudo da prevalência e bacteriologia das otites purulentas em suínos apresentando a SMDS, bem como em animais de baixo desenvolvimento e de crescimento normal. No total, foram examinados 385 suínos com idades entre 60 e 130 dias. De 242 animais com a SMDS, 57 (23,5%) apresentaram lesões purulentas no ouvido médio. Dentre 119 animais de baixo desenvolvimento, apenas 1 (0,7%) apresentou a lesão. Não foram detectadas lesões macroscópicas no ouvido médio dos 24 animais com crescimento normal (controles). Os agentes isolados com maior freqüência das lesões foram Arcanobacterium pyogenes, Streptococcus α– hemolíticos e Pasteurella multocida, encontrados em, respectivamente, 37 (43%), 32 (37,2%) e 24 (27,9%) dos 86 ouvidos submetidos à bacteriologia. A alta prevalência de lesões purulentas no ouvido médio de animais com a SMDS sugere que a infecção pelo PCV2 pode tornar o suíno mais suscetível às otites bacterianas. Por outro lado, a prevalência reduzida das lesões em suínos de baixo desenvolvimento sugere que a otite não representa uma causa importante de mau desempenho em suínos nas fases de crescimento e terminação. O isolamento do A. pyogenes, de Streptococcus α- hemolíticos e da P. multocida na maioria das lesões está de acordo com relatos anteriores, confirmando a importância desses organismos como agentes causais da otite média em suínos. / Postweaning Multisystemic Wasting Syndrome (PMWS) is an emerging disease disseminated globally that causes severe losses to the pig industry. Porcine circovirus type 2 (PCV2) is the causal agent of the disease and causes lesions mainly in lymphoid tissue and it is suggested that it can cause immunosuppression, predisposing the host to viral, bacterial and mycotic infections. In the present work we describe a study on prevalence and bacteriology of purulent otitis in pigs with PMWS, as well as in pigs with attrition and pigs with normal growth. A total amount of 385 animals were examined, with ages ranging from 60 to 130 days. Among 242 pigs with PMWS, 57 (23,5%) showed purulent lesions in the middle ear. Among 119 pigs with attrition, only 1 (0,7%) presented the lesion. In 24 control pigs, middle ear lesions were not detected. The agents most frequently isolated from the lesions were Arcanobacterium pyogenes, α–hemolytic Streptococci and Pasteurella multocida, found respectively in 36 (43%), 32 (37,2%) and 24 (27,9%) of 86 ears bacteriologically examined. The high prevalence of purulent lesions found in middle ear of PMWS affected pigs suggests that PCV2 infection can increase susceptibility of swine to bacterial otitis. On the other hand, the small prevalence of lesions in piglets with attrition suggests that otitis does not represent a significant cause for depressed growth in pigs from growing and finishing ages. The isolation of A. pyogenes, α-hemolytic Streptococci and P. multocida from most lesions agrees with previous reports, confirming the importance of these organisms as causal agents in the etiology of otitis media in pigs.

A Síndrome Multissistêmica do Definhamento do Suíno (SMDS) é uma doença emergente e mundialmente distribuída, que tem trazido sérios prejuízos econômicos para a indústria suinícola. O Circovírus Suíno tipo 2 (PCV2), agente causal da doença, provoca lesões principalmente nos tecidos linfóides, e sugere-se que produza imunossupressão, predispondo o hospedeiro a infecções virais, bacterianas e fúngicas secundárias. Neste trabalho, é descrito um estudo da prevalência e bacteriologia das otites purulentas em suínos apresentando a SMDS, bem como em animais de baixo desenvolvimento e de crescimento normal. No total, foram examinados 385 suínos com idades entre 60 e 130 dias. De 242 animais com a SMDS, 57 (23,5%) apresentaram lesões purulentas no ouvido médio. Dentre 119 animais de baixo desenvolvimento, apenas 1 (0,7%) apresentou a lesão. Não foram detectadas lesões macroscópicas no ouvido médio dos 24 animais com crescimento normal (controles). Os agentes isolados com maior freqüência das lesões foram Arcanobacterium pyogenes, Streptococcus α– hemolíticos e Pasteurella multocida, encontrados em, respectivamente, 37 (43%), 32 (37,2%) e 24 (27,9%) dos 86 ouvidos submetidos à bacteriologia. A alta prevalência de lesões purulentas no ouvido médio de animais com a SMDS sugere que a infecção pelo PCV2 pode tornar o suíno mais suscetível às otites bacterianas. Por outro lado, a prevalência reduzida das lesões em suínos de baixo desenvolvimento sugere que a otite não representa uma causa importante de mau desempenho em suínos nas fases de crescimento e terminação. O isolamento do A. pyogenes, de Streptococcus α- hemolíticos e da P. multocida na maioria das lesões está de acordo com relatos anteriores, confirmando a importância desses organismos como agentes causais da otite média em suínos. / Postweaning Multisystemic Wasting Syndrome (PMWS) is an emerging disease disseminated globally that causes severe losses to the pig industry. Porcine circovirus type 2 (PCV2) is the causal agent of the disease and causes lesions mainly in lymphoid tissue and it is suggested that it can cause immunosuppression, predisposing the host to viral, bacterial and mycotic infections. In the present work we describe a study on prevalence and bacteriology of purulent otitis in pigs with PMWS, as well as in pigs with attrition and pigs with normal growth. A total amount of 385 animals were examined, with ages ranging from 60 to 130 days. Among 242 pigs with PMWS, 57 (23,5%) showed purulent lesions in the middle ear. Among 119 pigs with attrition, only 1 (0,7%) presented the lesion. In 24 control pigs, middle ear lesions were not detected. The agents most frequently isolated from the lesions were Arcanobacterium pyogenes, α–hemolytic Streptococci and Pasteurella multocida, found respectively in 36 (43%), 32 (37,2%) and 24 (27,9%) of 86 ears bacteriologically examined. The high prevalence of purulent lesions found in middle ear of PMWS affected pigs suggests that PCV2 infection can increase susceptibility of swine to bacterial otitis. On the other hand, the small prevalence of lesions in piglets with attrition suggests that otitis does not represent a significant cause for depressed growth in pigs from growing and finishing ages. The isolation of A. pyogenes, α-hemolytic Streptococci and P. multocida from most lesions agrees with previous reports, confirming the importance of these organisms as causal agents in the etiology of otitis media in pigs.

As doenças respiratórias constituem um sério problema em sistemas intensivos de criação de suínos, causando enormes prejuízos à industria suína no Brasil e no mundo. Estes prejuízos estão freqüentemente relacionados à redução de peso, mortalidade, maior predisposição a doenças entéricas, gastos com vacinas e medicamentos. Os distúrbios respiratórios em suínos são manifestados através de um complexo de doenças, com envolvimento de agentes virais, bacterianos e fúngicos. Dentre estes, a presença do circovírus (PCV2) e o Pneumocystis sp. começa a ser gradativamente caracterizada como uma associação entre um agente causador de imunossupressão e um organismo de ação oportunista. O trabalho objetivou diagnosticar Pneumocystis sp. através das técnicas de imunohistoquímica, Grocott e nested-PCR, em suínos abatidos nos Estados do Rio Grande do Sul (RS) e Mato Grosso (MT), diagnosticar a ocorrência de PCV2 na mesma população de suínos, verificar a associação entre Pneumocystis sp. e PCV2, e determinar as relações filogenéticas entre as amostras de Pneumocystis sp. O estudo avaliou um total de 591 pulmões, 297 com alterações macroscópicas (pneumonia) e 294 normais obtidos em frigoríficos. Foram analisados 292 pulmões procedentes do RS e 299 pulmões do MT Para diagnóstico do Pneumocystis sp. as amostras foram analisadas através das técnicas de Grocott, Imunohistoquímica e nested-PCR (mtLSU e mtSSU rRNA). Do total das amostras, 36,9% foram positivas para Pneumocystis. O índice de positividade para o vírus PCV2 foi de 32,7% na amostra total. Os resultados revelaram uma alta prevalência do vírus (PCV2) em pulmões sem lesões macroscópicas. A co-infecção (PCV2 e Pneumocystis sp.), foi detectada em 28,0% em 564 pulmões examinados. As análises das seqüências dos nucleotídeos dos produtos de PCR dos genes mtLSU e mtSSU do rRNA do Pneumocystis sp. nos pulmões analisados, sugerem a presença até o presente momento de 2 espécies diferentes de Pneumocystis no Brasil. Este estudo evidencia a ocorrência da co-infecção de dois agentes (Pneumocystis sp. e PCV2) em animais hígidos, fato que, indica a necessidade de planejamento e implementação de medidas de controle para melhorar a produtividade na suinocultura. / Respiratory diseases are a major problem in intensive systems of swine husbandry. They are a cause for high losses in the swine industry in Brazil and in the world. These losses are often related to weight reduction, mortality, higher vulnerability to enteric diseases, and expenses with vaccines and drugs. Respiratory diseases in swine appear through a complex of diseases, caused by virus, bacteria and fungi; among these, the porcine circovirus 2 (PCV2) and Pneumocystis sp., the former an agent which causes immunosupression and the latter an oportunistic microorganism. Both are being recognized as capable of being associated. The objectives of this study were to: identify Pneumocystis sp. through immunohystochemistry techniques, Grocott and nested-PCR in swine slaughtered in the States of Rio Grande do Sul and Mato Grosso (MT); investigate PCV2 in the same swine population; investigate the association between Pneumocystis sp. and PCV2, and establish a filogenetic relationship between isolated of Pneumocystis sp. The study was carried out with a total of 591 lungs, 297 with macroscopic alterations characteristic of pneumonia, and 294 normal lungs from the industry. 292 lungs came from RS and 299 lungs came from MT. In order, to diagnose Pneumocystis infection, samples were analysed through Grocott technique, immunohystochemistry and nested-PCR (mtLSU and mtSSU rRNA). Among all samples 36,9% were positive for Pneumocystis sp.. PCV2 virus was found in 37,2% of the samples. Results revealed a high prevalence of the PCV2 virus in lungs without macroscopic lesions. Co-infection (PCV2 and Pneumocystis sp.) was found in 28,0% of 564 lungs examined. So far, the analyses of the sequences of nucleotides from the products of PCR from the genes mtlSU rRNA and mtSSU rRNA from Pneumocystis obtained from the examined lungs suggest that, it is possible the existence of two different species of Pneumocystis in Brazil. This study shows co-infection by two agents (Pneumocysts sp. and PCV2) in apparently healthy animals. This fact points out the necessity planning and implementation of control measures in order to improve productiviy in swine husbandry and industry.

Respiratorische Krankheitsbilder, an denen mehr als ein Pathogen ursächlich beteiligt ist, gewinnen in der Schweinepopulation zunehmend an Bedeutung. Diagnostische Methoden zum simultanen Nachweis mehrerer Erreger sind Bestandteil einer schnellen und effizienten Therapie und tragen zum ökonomischen Bestandsmanagement bei. Im Rahmen dieser Arbeit sollten Methoden für den Multiplex-Nachweis von Antikörpern und Nukleinsäuren viraler Erreger von respiratorischen Krankheitsgeschehen des Schweins entwickelt werden. Die Methode für den Multiplex-Nachweis von Antikörpern sollte auf Basis der xMAP® Flüssigchip-Technologie (Luminex Corporation, Austin, T, USA) an der LiquiChip®- Workstation (Qiagen, Hilden, D) etabliert werden. Da es sich um eine für den Antikörpernachweis im veterinärmedizinischen Bereich bislang nicht genutzte Methode handelte, erfolgte die Prüfung der Machbarkeit zunächst im Einfach-Format am Beispiel des Porzinen Circovirus Typ 2. Im Laufe der Arbeit wurde deutlich, daß die Kopplung des PCV2 ORF2-Proteins als Capture-Molekül sowie die Erstellung der Versuchsansätze mit akzeptablem Aufwand ohne Spezialtechniken durchführbar war. Aufgrund der Anordnung der Proben auf Platten im 96-well-Format und der vollautomatischen Messung war ein hoher Probendurchsatz möglich. Nach der Einführung von Waschschritten in die Versuchsansätze konnten hohe Fluoreszenzsignale erzeugt werden. Im Laufe der Optimierungsversuche wurde allerdings die fehlende Korrelation dieser Fluoreszenzsignale mit den Ergebnissen der Referenzmethode deutlich. Aufgrund der unbekannten Testeigenschaften sowie fehlender Kontrollmöglichkeiten wurden diese nicht sogleich als unspezifische bzw. falsch positive Signale erkannt. Erst durch die Testung von positiven und negativen Feldseren an verschiedensten Bead-Arten wurde ersichtlich, daß die Fluoreszenzen nicht ausschließlich durch die spezifische Bindung der PCV2-Antikörper an das Capture-Protein entstanden. Im Ausschlussverfahren konnte die Ursache eingegrenzt werden. Bestandteile aus dem Schweineserum führten vermutlich durch unspezifische Bindungen an die LiquiChip®-Beads zu einem Fluoreszenzereignis. Durch Vorinkubation der Beads in Pferdeserum und der Feldseren mit einem Block-Puffer wurde versucht, diese Serumbestandteile abzusättigen und so eine Bindung an die Beads zu verhindern. Die Inkubationsvarianten führten weder zu einer Minimierung der unspezifischen Bindung noch zu einer verbesserten Differenzierung PCV2-positiver und negativer Seren. Die in der vorliegenden Arbeit verwendeten Bead-Arten sind für den Nachweis von Antikörpern gegen das PCV2 ORF2-Protein nicht geeignet. Alternative Bead-Arten für einen vergleichbaren Versuchsansatz stehen derzeit nicht zur Verfügung. Ein weiteres Ziel der Arbeit bestand darin, eine bereits in der Diagnostik von Schweineviren etablierte Methode, die PCR, zu einer Multiplex-PCR zu erweitern. Als zu detektierende Parameter wurden die derzeit bedeutendsten viralen Erreger von respiratorischen Erkrankungen des Schweins, das PRRS-Virus (Typ 1 und Typ 2), das Porzine Influenzavirus mit den Subtypen H1N1, H3N2 und H1N2 und PCV2 gewählt. Es wurden die Primersequenzen von bereits etablierten Einfach-PCRs an die besonderen Ansprüche einer Multiplex-PCR angepasst und die Methode zunächst im Einzelansatz auf Funktionsfähigkeit überprüft. Im Anschluss wurden die Parameter zu einer Multiplex-PCR zusammengeführt, die Methode optimiert und auf Spezifität, Sensitivität und Verhalten in der Routinediagnostik überprüft. Aufgrund der im Gegensatz zur Einfach-PCR zum Teil herabgesetzten Sensitivität ist diese Methode für Ausschlussuntersuchungen weniger geeignet. Für die Untersuchung von Probenmaterial klinisch erkrankter Tiere ist sie jedoch gut geeignet und bietet die Möglichkeit einen schnellen Überblick über das Erregerspektrum zu erhalten. Es muss jedoch berücksichtigt werden, daß bestimmte Parameter, z.B. PCV2, die Sensitivität des Nachweises der anderen Parameter sehr deutlich herabsetzen kann. Dies ist insbesondere von Bedeutung, da PCV2-DNA in Probenmaterial von klinisch erkrankten Tieren in sehr hohen Mengen vorhanden ist und dadurch die weiteren Parameter noch zusätzlich beeinflusst werden können.

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), whereas the ubiquitous porcine circovirus type 1 (PCV1) is nonpathogenic for pigs. Since its initial detection in a Canadian commercial swine herd in 1991, PMWS has been detected in all swine producing regions of the world and is now a serious economic problem to the swine industry. The objectives of this dissertation were to biologically, genetically and experimentally characterize both PCV1 and PCV2, to identify the genetic determinant(s) for virulence and replication, and to develop an effective genetically-engineered vaccine against PCV2 infection and PMWS. The genetic heterogeneity of PCV2 and PCV1 isolates from different geographic origins were determined. We found that, although PCV1 and PCV2 genomes were very conserved, some minor genomic variation exists among PCV1 isolates and PCV2 isolates. The nonpathogenic PCV1 and pathogenic PCV2 share only about 76% nucleotide sequence identity but have similar genomic organization. The highest sequence variability among PCV isolates is found in the immunogenic ORF2 capsid gene. Based on the sequence data in this dissertation, a universal polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was developed that is capable of detecting all known PCV isolates and differentiating between infections by nonpathogenic PCV1 and pathogenic PCV2. In order to study the structural and functional relationship of PCV genes and to develop a genetically-engineered vaccine, we constructed infectious DNA clones of both PCV1 and PCV2. By using the PCV2 infectious clone, we showed that pigs can be infected by direct intrahepatic injection of PCV2 infectious DNA clone. The pathological lesions and clinical disease associated with PCV2 infection were more definitively characterized by using the infectious DNA clone. We found that PCV2 is the primary but not the sole causative agent of PMWS, as the full spectrum of clinical PMWS was not reproduced by the infectious PCV2 DNA clone although pathological lesions characteristic of PMWS were reproduced. A chimeric vaccine was constructed by cloning the immunogenic capsid gene of the pathogenic PCV2 into the genomic backbone of the non-pathogenic PCV1 virus. We showed that the resulting chimeric PCV1-2 vaccine virus, retained the non-pathogenic nature of PCV1 but induced a protective immune response against a wild-type PCV2 challenge. In vaccinated pigs, the chimeric PCV1-2 vaccine reduced PCV2 viremia length and serum virus loads and reduced pathological lesions such as lymphoid depletion (LD) and histiocytic replacement (HR) in lymphoid tissues, inflammation and discoloration of the lymph nodes. The amounts of PCV2 antigen and PCV2 genomic copy loads in lymph node tissues were also significantly reduced. Our results indicated that the attenuated chimeric PCV1-2 virus induces protective immunity against PCV2 infection and thus could serve as an effective vaccine against PCV2 and PMWS. To improve the safety of the vaccine, we attempted to identify the genetic determinant(s) for PCV2 virulence. An isolate of PCV2 was serially passaged for 120 times in PK-15 cells. After 120 passages, a total of two amino acid mutations were identified in the capsid protein of the passage 120 virus (VP120), P110A and R191S. Compared to other known PCV1 and PCV2 sequences, the two amino acid mutations in PCV2 VP120 are unique. The VP120 virus was biologically characterized in vitro and experimentally characterized in specific-pathogen-free (SPF) pigs. The two amino acid mutations resulted in an enhanced replication ability of PCV2 VP120 in PK-15 cells and an attenuated phenotype in infected pigs. The P110A and R191S mutations in the capsid protein either alone or collectively are likely important for PCV2 virulence and replication. In summary, we genetically characterized PCV2 isolates from different geographic regions and developed a PCR-RFLP assay. We constructed and characterized infectious DNA clones of PCV1 and PCV2, and developed a genetically engineered vaccine against PCV2 infection. We also identified the genetic determinants for PCV2 virulence and replication. The vaccine developed in this study, when it becomes available, will help the swine industry control this important pathogen. / Ph. D.

No description available.

Made available in DSpace on 2016-12-08T16:24:07Z (GMT). No. of bitstreams: 1 PGCA09MA052.pdf: 726948 bytes, checksum: 2a141d4d92b1c5ac552655f7c9ad3c1a (MD5) Previous issue date: 2009-11-06 / Many infectious agents have been associated with reproductive failure in swine, representing significantly economic losses for production. Recently, Porcine Circovirus Type 2 (PCV2), etiologic agent of PCVAD or PCV2 associated diseases, was associated with reproductive failure in swine around the world. To confirm the pathogenic potential of PCV2 inducing reproductive failure in sows, it s necessary the viral isolation and antigen and nucleic acid demonstration in fetuses. Other viral agent, Torque Teno Vírus (TTV), also have been recently associated with infections caused by PCV2. TTV alone has not showed pathogenic signals in swine, but, its role in co-infections with other pathogens has been investigated. The present study aimed the diagnostic of PCV2 in natural infections where there was reproductive failure, as well as to establish and apply the Polimerase Chain Reaction (PCR) technique for TTV from organs. Samples from field cases, as aborted fetuses, mummified, stillborn, fragile piglets and material from abattoir sows were collected and processed to diagnostic infection in order to detect PCV2 by PCR and immunohistochemistry (IHC). Samples were collected from 21 farms; and a total of 169 fetuses were necropsied. Moreover, reproductive samples from 83 abattoir sows were collected in 4 slaughterhouses of Santa Catarina State. In the present study was possible detect viral DNA by PCR in 29 (17,1%) of 169 analyzed fetuses, where heart and lymphoid tissues showed virus DNA more frequently, 41,4% and 37,8%, respectively. Viral presence was confirmed by IHC in tissues, which detected viral antigens in 17 PCV2 positives fetuses by PCR. Samples of reproductive tissues from sows also were tested by PCR and PCV2 was identified in 4 sows (4,8%). PCR technique aimed to detect TTV was established for viral DNA from organs. Samples of reproductive tissues from sows were tested, and were found both genogroups of TTV (TTV1 and TTV2), in 25 (30,1%) and 41 (49,3%) sows, respectively. Fetuses samples that resulted positive to PCV2 by PCR were also tested to TTV, and it was observed the occurrence of co-infection between these agents. The results obtained here suggest the involvement of PCV2 in reproductive failure in sows, besides show that TTV was present in analyzed samples, corroboring the association with PCV2 / Muitos agentes infecciosos têm sido associados às falhas reprodutivas na produção de suínos, representando significativas perdas econômicas para os suinocultores. Recentemente o Circovirus Suíno tipo 2 (PCV2), agente etiológico da circovirose suína, foi associado a falhas reprodutivas em suínos em diversas partes do mundo. Para confirmar o potencial patogênico do PCV2 causando falhas reprodutivas em porcas, é necessário o isolamento do vírus e demonstração de antígeno e ácido nucléico viral em fetos. Outro agente viral, o Torque Teno Vírus (TTV), também foi recentemente associado às infecções causadas pelo PCV2. O TTV sozinho ainda não tem se mostrado patogênico em suínos, porém, seu papel em co-infecções com outros patógenos vem sendo investigado. O presente trabalho teve por objetivos diagnosticar o PCV2 em infecções naturais onde existiam falhas reprodutivas, assim como padronizar e aplicar a técnica de Reação em Cadeia da Polimerase (PCR) para TTV a partir de órgãos. Amostras provenientes de casos clínicos de campo, como fetos abortados, mumificados, natimortos, leitões inviáveis e material de fêmeas descartadas foram coletadas e processadas para diagnóstico da infecção pelo PCV2 através de PCR e imunoistoquímica (IHQ). Foram colhidas amostras de 21 granjas produtoras de suínos, totalizando 169 fetos, que foram necropsiados para coleta de órgãos. Além disso, amostras de órgãos reprodutivos de 83 fêmeas descartadas foram colhidas em 4 abatedouros da região oeste catarinense. No presente estudo foi possível detectar DNA viral por PCR em 29 (17,1%) dos 169 fetos analisados, sendo coração e tecidos linfóides os órgãos onde o vírus foi identificado com maior freqüência, 41,4% e 37,8%, respectivamente. A presença do vírus foi confirmada por teste de IHQ dos tecidos, sendo encontrado antígeno viral em 17 fetos positivos para PCV2 por PCR. As amostras de tecido reprodutivo das fêmeas também foram testadas por PCR e o PCV2 foi identificado em 4 porcas (4,8%). Visando a detecção de TTV foram testadas por PCR amostras de órgãos reprodutivos de fêmeas suínas, sendo diagnosticados os dois genogrupos de TTV, TTV1 e TTV2 em 25 (30,1%) e 41 (49,3%) fêmeas, respectivamente. As amostras de fetos que resultaram positivas para PCV2 pela técnica de PCR também foram testadas para TTV, observando-se a ocorrência de coinfecção entre estes agentes. Os resultados obtidos evidenciam o provável envolvimento do PCV2 em falhas reprodutivas em fêmeas suínas, bem como mostram que o TTV está presente nas amostras analisadas, confirmando a associação com o PCV2

Porcine circovirus type 2 (PCV2) is the causative agent of porcine circovirus-associated disease (PCVAD). Porcine reproductive and respiratory syndrome (PRRS) is caused by PRRS virus (PRRSV). Both PCV2 and PRRSV have caused devastating diseases in the swine industry worldwide, resulting in immense economic losses. One of the most common co-infections in the swine industry is PCV2 and PRRSV. The aim of this dissertation research is to explore different experimental approaches to develop novel vaccines against the two major pathogens affecting swine production and study the basic mechanisms that may be involved in viral pathogenesis. Two types of porcine circovirus (PCV), PCV1 and PCV2, have been identified thus far. PCV1, first identified as a contaminant of the PK-15 cell line, is non-pathogenic and has a low prevalence in swine herds. PCV2 is highly prevalent in most swine-producing countries and is associated with clinical PCVAD. The non-pathogenic PCV1 shares similar genomic organization with PCV2. Previously, it has been demonstrated that a genetically modified infectious chimeric PCV1-2a virus can tolerate up to a 27 aa insertion in the C-terminus of the ORF2 without affecting infectivity and produce a dual immune response against PCV2cap and the inserted epitope tag. Therefore, we evaluated the use of the non-pathogenic PCV1 wild-type (wt) virus and chimeric PCV1-2a vaccine virus (vs) to express four known B-cell epitopes of PRRSV. Peptide epitopes of PRRSV-VR2385, including GP2II (aa 40–51, ASPSHVGWWSFA), GP3I (aa 61–72, QAAAEAYEPGRS), GP5I (aa 35–46, SSSNLQLIYNLT), and GP5IV (aa 187–200, TPVTRVSAEQWGRP) were inserted in frame into the C-terminus of the ORF2 of PCV1wt as well as the PCV1-2avs. Four PCV1-PRRSVEPI chimeric viruses and four PCV1-2a-PRRSVEPI chimeric viruses were successfully rescued and shown to be infectious in vitro and co-expressed PCV1cap or PCV2cap with each specific PRRSV epitope. Two independent animal studies were conducted to evaluate whether the non-pathogenic PCV1 can serve as a vaccine delivery vector and whether the PCV1-2a vaccine virus can be used to develop a bivalent vaccine against both PCV2 and PRRSV. We demonstrated that three PCV1-PRRSVEPI chimeric viruses and two PCV1-2a-PRRSVEPI chimeric viruses were infectious in pigs. Importantly, we demonstrated that the PCV1-PRRSVEPI and PCV1-2a-PRRSVEPI chimeric viruses not only induced specific PCV1 or PCV2 IgG antibody but also specific anti-PRRSV epitope antibody responses as well. Regardless of the PCV backbone used, we showed that the PCV-PRRSV chimeric viruses elicited neutralizing antibodies against PRRSV-VR2385. These results provided a proof of concept for the potential use of the non-pathogenic PCV1 as a vaccine delivery system for PRRSV or other swine pathogens and the use of PCV1-2a vaccine virus to generate a bivalent vaccine against both PCV2 and PRRSV. PRRSV causes a persistent infection and immunosuppression. Immunomodulation of the host immune system is caused by modulation of numerous interleukins, such as type I interferons, tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), interleukin-6 (IL-6), and interleukin-12 (IL-12) in infected pigs. Antigen-presenting cells (APCs) are the first line of defense, and their infection plays an important role in innate-mediated immune regulation during early immune responses. Among the APCs, pulmonary alveolar macrophages (PAMs), pulmonary interstitial macrophages (PIMs), and dendritic cells (DCs) are the main targets for PRRSV replication. The role of PRRSV-DCs interaction is not fully understood, and current research focuses on the production and regulation of interferons through DC-SIGN receptors. In this study, we evaluated the immunomodulation of MoDCs by PRRSV through interactions with the pDC-SIGN receptor, by blocking pDC-SIGN with recombinant hICAM-3-Fc or anti-pDC-SIGN mAb. Our results indicate that recombinant hICAM-3-Fc enhances mRNA expression of proinflammatory cytokines and that anti-pDC-SIGN mAb inhibits mRNA expression of TNF-α and IL-1α and enhances the expression of IL-12 induced by PRRSV in MoDCs. The results will help understand the molecular mechanisms of PRRSV pathogenesis. / Ph. D.