Orientador: João Pessoa Araujo Junior / Resumo: A suinocultura vem alcançando grande desenvolvimento de técnicas eficientes associadas ao melhoramento genético, nutrição, manejo e sanidade. Entretanto, devido aos métodos intensivos de criação, os suínos se tornaram mais susceptíveis a um grande número de doenças infecciosas. Entre os mais importantes patógenos que afetam a indústria suinícola mundial está o circovírus suíno 2 (PCV2), um pequeno vírus icosaédrico, não-envelopado, de DNA circular, de fita simples (ssDNA), ambisenso, composta por 1767-1768 nucleotídeos. Este vírus é altamente resistente a variações ambientais e agentes desinfetantes, é endêmico no mundo todo e está associado a várias manifestações clínicas distintas, que acarretam importantes perdas econômicas aos produtores. Um dos fatores possivelmente implicados na patogenicidade do PCV2 é a proteína Cap, a unidade fundamental constituinte do capsídeo deste vírus. Estudos realizados pela equipe do Prof. Dr. João Pessoa Araújo Jr., do Instituto de Biotecnologia da Unesp em Botucatu/SP, comprovaram que vírus com mutações em suas proteínas Cap isolados a partir de cultivo celular aumentavam a morte celular em culturas celulares infectadas. Estes resultados evidenciaram a importância do capsídeo nos mecanismos de infecção e patogenicidade do PCV2, tornando interessante a realização de estudos estruturais com as proteínas Cap mutantes. A execução de estudos estruturais in silico mostrou a baixa frequência das mutações identificadas na proteína Cap dos vírus pro... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Swine breeding has achieved a high development based on genetic improvement, nutrition, management and sanity. However, due to the intensive breeding methods, swine have become more susceptible to a higher number of infectious diseases. Among the most important pathogens that affect the swine world industry is the porcine circovirus 2 (PCV2), a small, icosahedral, non-enveloped virus, ambisense single-stranded circular DNA, composed by 1,767-1,768 nucleotides. This virus is highly resistant to environmental variations and disinfecting agents, endemic worldwide and has been associated to several distinct clinical manifestations that entail important economic losses to the producers. One of the factors possibly implicated on the PCV2 pathogenicity is the Cap protein, the fundamental unity that constitutes this virus capsid. Studies performed by the group of Dr. João Pessoa Araújo Jr. rom the Instituto de Biotecnologia da Unesp em Botucatu/SP, confirmed that viruses with mutated Cap proteins from cell culture increased cell death in infected cultures. Such results highlight the importance of capsid in the infection mechanisms and pathogenicity of PCV2 and the importance of structural and comparative studies with Cap protein structures. In silico structural studies showed the low frequency of the mutations identified in the mutant Cap proteins and also indicated a clear difference between the physico-chemical properties of the new amino acid residues in comparison to those found ... (Complete abstract click electronic access below) / Mestre

Submitted by Pâmela Merlo Marson (pam.marson@aluno.ibb.unesp.br) on 2018-06-05T17:11:43Z No. of bitstreams: 1 Dissertação_Mestrado_Pamela-Merlo-Marson_2018_vf.pdf: 1757229 bytes, checksum: ed7b8faed4d956e7a1ba89178199507d (MD5) / Approved for entry into archive by Sulamita Selma C Colnago null (sulamita@btu.unesp.br) on 2018-06-07T14:10:28Z (GMT) No. of bitstreams: 1 marson_pm_me_bot.pdf: 1757229 bytes, checksum: ed7b8faed4d956e7a1ba89178199507d (MD5) / Made available in DSpace on 2018-06-07T14:10:28Z (GMT). No. of bitstreams: 1 marson_pm_me_bot.pdf: 1757229 bytes, checksum: ed7b8faed4d956e7a1ba89178199507d (MD5) Previous issue date: 2018-02-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A suinocultura vem alcançando grande desenvolvimento de técnicas eficientes associadas ao melhoramento genético, nutrição, manejo e sanidade. Entretanto, devido aos métodos intensivos de criação, os suínos se tornaram mais susceptíveis a um grande número de doenças infecciosas. Entre os mais importantes patógenos que afetam a indústria suinícola mundial está o circovírus suíno 2 (PCV2), um pequeno vírus icosaédrico, não-envelopado, de DNA circular, de fita simples (ssDNA), ambisenso, composta por 1767-1768 nucleotídeos. Este vírus é altamente resistente a variações ambientais e agentes desinfetantes, é endêmico no mundo todo e está associado a várias manifestações clínicas distintas, que acarretam importantes perdas econômicas aos produtores. Um dos fatores possivelmente implicados na patogenicidade do PCV2 é a proteína Cap, a unidade fundamental constituinte do capsídeo deste vírus. Estudos realizados pela equipe do Prof. Dr. João Pessoa Araújo Jr., do Instituto de Biotecnologia da Unesp em Botucatu/SP, comprovaram que vírus com mutações em suas proteínas Cap isolados a partir de cultivo celular aumentavam a morte celular em culturas celulares infectadas. Estes resultados evidenciaram a importância do capsídeo nos mecanismos de infecção e patogenicidade do PCV2, tornando interessante a realização de estudos estruturais com as proteínas Cap mutantes. A execução de estudos estruturais in silico mostrou a baixa frequência das mutações identificadas na proteína Cap dos vírus provenientes do cultivo in vitro e também indicou uma clara diferença entre as propriedades físico-químicas dos novos resíduos de aminoácidos em relação àqueles substituídos. Estas alterações, associadas à localização dos resíduos na superfície viral e a menor flexibilidade das proteínas Cap dos vírus mutantes, indicaram a possibilidade de alterações estruturais/funcionais relevantes, incluindo a alteração da afinidade por receptores celulares e diminuição da efetividade de anticorpos produzidos contra vírus vacinais. Foram também realizados trabalhos experimentais para a produção heteróloga da proteína Cap de um vírus selvagem, os quais envolveram ensaios de subclonagem da sequência de interesse em um vetor de expressão, testes de transcrição e experimentos de expressão protéica. Os resultados destes procedimentos foram compatíveis com a produção da proteína Cap, porém novos estudos são necessários para confirmar a produção da molécula alvo e melhorar o rendimento dos ensaios de expressão. / Swine breeding has achieved a high development based on genetic improvement, nutrition, management and sanity. However, due to the intensive breeding methods, swine have become more susceptible to a higher number of infectious diseases. Among the most important pathogens that affect the swine world industry is the porcine circovirus 2 (PCV2), a small, icosahedral, non-enveloped virus, ambisense single-stranded circular DNA, composed by 1,767-1,768 nucleotides. This virus is highly resistant to environmental variations and disinfecting agents, endemic worldwide and has been associated to several distinct clinical manifestations that entail important economic losses to the producers. One of the factors possibly implicated on the PCV2 pathogenicity is the Cap protein, the fundamental unity that constitutes this virus capsid. Studies performed by the group of Dr. João Pessoa Araújo Jr. rom the Instituto de Biotecnologia da Unesp em Botucatu/SP, confirmed that viruses with mutated Cap proteins from cell culture increased cell death in infected cultures. Such results highlight the importance of capsid in the infection mechanisms and pathogenicity of PCV2 and the importance of structural and comparative studies with Cap protein structures. In silico structural studies showed the low frequency of the mutations identified in the mutant Cap proteins and also indicated a clear difference between the physico-chemical properties of the new amino acid residues in comparison to those found in the wild-type virus. These mutations, associated with the location of the mutated residues on the viral surface and the lower mutated Cap protein flexibility, could lead to relevant structural/functional changes, including alteration of affinity for cellular receptors and decreased effectiveness of antibodies produced against vaccine viruses. Experimental works aiming the heterologous production of a wild-type Cap protein were also carried out, which involved expression vector subcloning, transcription tests and protein expression experiments. The results of these procedures were compatible with the production of the Cap protein, but further studies are needed to confirm the production of the target molecule and improve the yield of the expression assays.

Made available in DSpace on 2015-03-26T13:46:51Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1965376 bytes, checksum: 32df14418e8fde9c7b4067ac9f8ea6c6 (MD5) Previous issue date: 2010-05-07 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Porcine circovirus 2 (PCV-2) is the major causative agent of postweaning multisystemic wasting syndrome (PMWS) and is associated with different syndromes that affect the swine. The term PCVAD (Porcine circovirus associated diseases) was introduced in 2006 to gather many diseases. Since serological studies are essential for monitoring the virus, the aim of this study was to develop enzyme immunoassays (ELISA) for detection and titration of antibodies to PCV-2 and also for viral quantification as well as the recombinant protein capsid PCV-2 (rCAP PCV-2) in extracts of Escherichia coli. After the standardization of the technique, 29 negative serum samples in ELISA were used for determining the cutt off. The quantitative ELISA was compared with the Immunoperoxidase monolayer (IPMA), through analysis of 155 serum samples, pre-determined by IPMA, of which 125 samples were positive and 30 negative samples for antibodies to PCV-2, in ELISA. The tests showed a concordance of 98.70% confirming the high sensitivity and specificity of ELISA on the IPMA. In the quantitative assay, 20 serum samples had the title determined by ELISA and it showed a higher detection limit of antibodies than the IPMA in 18 of the 20 samples tested. To quantify the rCAP PCV-2 in extracts of E.coli, a capture ELISA was developed. Were used precipitated IgGs from polyclonal rabbit serum anti-rCap PCV-2 as capture antibody and the conjugated IgGs with peroxidases as detection antibody. A standard curve was obtained by serial dilution of rCap PCV-2 and the test showed a detection limit in the range of 0.625 to 0.0097 mg/mL, which was successfully used to quantify the protein in the extract of E.coli. Thus it is expected that this test is also applied in the quantification of future PCV-2 in field samples. / O Circovírus suíno 2 (PCV-2) é o principal agente causador da Síndrome Multissistêmica do Definhamento dos Suínos (SMDS), e está associado a diferentes síndromes que acometem esses animais. O termo PCVAD (Porcine circovírus 2 associated diseases) foi introduzido em 2006, para enquadrar todas essas doenças. Uma vez que estudos sorológicos são fundamentais para o monitoramento do vírus, o objetivo desse trabalho foi desenvolver ensaios imunoenzimáticos (ELISA) para a detecção e titulação de anticorpos anti-PCV-2 e também para a quantificação viral, assim como da proteína recombinante do capsídeo do PCV-2 (rCap PCV-2) em extratos de Escherichia coli . Após a padronização da técnica, 29 amostras de soro negativos por ELISA indireto foram utilizadas para a determinação do cutt off. O ELISA qualitativo foi comparado com a Imunoperoxidase em Monocamada (IPMA), através da análise de 155 amostras de soro, pré-determinadas por IPMA, sendo 125 amostras positivas e 30 amostras negativas para anticorpos anti-PCV-2, no ELISA. Os testes apresentaram uma concordância de 98,70 % confirmando assim a alta sensibilidade e especificidade do ELISA relativa ao IPMA. No ensaio quantitativo, 20 amostras de soro tiveram o título determinado através do ELISA e este apresentou um maior limite de detecção de anticorpos do que o IPMA, em 18 das 20 amostras avaliadas. Para a quantificação da rCap PCV-2 presente em extratos de E.coli, um ELISA de captura foi desenvolvido. Foram utilizadas IgGs precipitadas a partir de soro policlonal de coelho anti-rCap PCV-2 como anticorpo de captura e as IgGs conjugadas com peroxidase como anticorpo de detecção. Uma curva-padrão foi obtida através da diluição seriada da rCap PCV-2 e o ensaio apresentou um limite de detecção na faixa entre 0,625 a 0,0097 μg/mL, que foi utilizado com sucesso para quantificar a proteína no extrato de E.coli. Assim espera-se que este teste seja também aplicado futuramente na quantificação do PCV-2 em amostras de campo.

Made available in DSpace on 2015-03-26T13:47:24Z (GMT). No. of bitstreams: 1 resumo.pdf: 759656 bytes, checksum: 08559ba05ec99261b3eaa0c86a4aa9e9 (MD5) Previous issue date: 2009-02-27 / Samples of mandibular, mediastinic, gastric, mesenteric and inguinal lynph nodes and lungs. kidneys, liver, heart and spleen of 24 pigs showing clinical features of postweaning multisystemic wasting syndrome (PMWS) and samples of inguinal lynph nodes of 24 clinicaly healthy pigs were colected. In one part of the samples were performed real time PCR for detection of the Porcine Circovirus 2 (PCV 2) nucleic acid. The other part were analysed for histopathological lesions. One part of the lynph nodes were submited to immunohistochemistry against the PCV 2 and this results were compared to the results of real time PCR Lynphocellular depletion and infiltration of histiocytic cells were the most seen lesions in lynph nodes. Based on the intensity of this lesions, three stages of the desease were determined Stage 1, mild lesions (39%), stage 2, moderate lesions (32,7%) and stage 3, severe lesions (20%). In the lungs, the majority of the lesions seen were intersticial pneumonia with lynphoid and hystiocytic infiltration of the alveolar septae ln the kidneys, intersticial nephritis with mononuclear infnltration were seen in 35% of the samples and glomerulonephritis with glomerular fibrin and neutrophill infiltration were seen in 50% of the samples. In the Iiver, lynphoid infiltration in the periportal zones were the lesions most observed. Lynphocellular depletion were obseved in the most samples of the spleen. No lesions were observed in the heart. Only 65.3% of the lynph nodes submitted to immunohistochemistry were considered positive for the presence of the PCV 2. Though 100% of the samples submitted to the real time PCR were considered positive for the presence of the virus. / Foram coletadas amostras de Iinfonodos mandibulares, mediastínicos, epigástricos, mesentéricos e inguinais superficiais, além de pulmões, rins, fígado. coração e baço de 24 suínos exibindo sinais clínicos da síndrome do definhamento multissistêmico pós-desmame (PMWS) e amostras de Iinfonodos inguinais superficiais de 24 suínos cIinicamente sadios. Uma pane dos tecidos foi encaminhada para a realização de PCR em tempo real para detecção do ácido nucléico do Circovírus suíno 2 (PCV 2). Outra parte das amostras foi incluída em parafina e cortada na espessura de 4 um para avaliação histopatológica. Uma parte dos linfonodos foi submetida ao teste de imunoistoquímica e este resultado foi comparado ao resultado do PCR em tempo real. Depleção Iinfóide e infiltrado macrofágico foram as lesões mais observadas nos linfonodos. Baseado na intensidade destas lesões foram determinados trés estágios da doença. Estágio 1, lesões discretas (39%), estágio 2, lesões moderadas (32,7%) e estágio 3, lesões graves (20%). Nos pulmões, a lesão mais encontrada foi pneumonia intersticial com espessamento dos septos alveolares por infltrado inflamatório mononuclear, principalmente linfócitos e histiócitos. Nos rins, a nefrite intersticial com infiltrado de linfócitos e histiócitos na medula e na córtex renal foi observada em 35 % das amostras e glomerulonefrite com infiltrado de fibrina e neutrófilos nos glomérulos foi observada em 50 % das amostras. No fígado, o achado mais freqüente foi infiltrado inflamatório linfocítico nas regiões periportais. Depleção linfóide foi observada na maioria das amostras de baço. Não foram observadas lesões nas amostras de coração. Apenas 65,3 % dos linfonodos submetidos à imunoistoquímica foram considerados positivos para a presença do PCV 2. Entretanto, 100°/o das amostras foram consideradas positivas para a presença do vírus pelo PCR em tempo real.

Porcine circovirus associated disease (PCVAD) is an umbrella term for a multitude of diseases and syndromes that have a negative impact on the health and economics of pig production operations throughout the world. Porcine circovirus type 2 is the causative agent of PCVAD; however the presence of PCV2 alone is rarely enough to cause clinical disease. In order for the full development of PCVAD the presence of a co-infecting pathogen is required. The mechanisms by which co-infection leads to disease remain ongoing areas of research, but it is thought that host immune modulations by PCV2 or a co-infecting pathogen are critical in the pathogenesis of PCVAD. In the first study of this dissertation the ability of PCV2 to induce regulatory T-cells (Tregs) and alter cytokine production was evaluated in vivo. The addition of PCV2 to a multiple viral challenge resulted in a significant increase in Tregs. Levels of IL-10 and IFN-γ were also found to be altered when PCV2 was added to a multiple viral challenge. In further experiments, monocyte derived dendritic cells (MoDC) were infected with different combinations and strains of PCV2 and PRRSV in vitro and evaluated for expression levels of programmed death ligand-1 (PD-L1), IL-10, CD86, swine leukocyte antigen-1 (SLA-1), and swine leukocyte antigen-2 (SLA-2). Expression levels of PD-L1 were significantly increased in PCV2 and PRRSV co-infected MoDCs. SLA-1, SLA-2, and CD86 expression levels were significantly decreased in the MoDC treatment groups containing both PCV2 and virulent stains of PRRSV. MoDC IL-10 expression was significantly increased by PCV2 and virulent strains of PRRSV co-infection. Finally, we investigated the role of the PD-L1/programmed death ligand-1 (PD-1) axis in porcine lymphocyte anergy, apoptosis, and the induction of Tregs. Lymphocyte populations with normal PD-1 expression had significantly higher percentages of anergic and apoptotic lymphocytes, and CD4+CD25HighFoxP3+ Tregs when compared to a PD-1 deficient lymphocyte population. The findings from these studies indicate host immune modulation by PCV2 in vivo and the development of a regulatory phenotype of dendritic cell following PCV2/PRRSV co-infections in vitro that may contribute to a dysfunctional adaptive immune response and the overall pathogenesis of PCVAD. / Ph. D.

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.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Steven S. Dritz / Vaccination against porcine circovirus type 2 (PCV2) has become a standard practice to improve pig mortality and growth rate in PCV2-affected herds. Unfortunately, there has been little field-based research evaluating factors which affect circovirus vaccination. The focus of this research was on potential vaccination-affecting factors such as age, dosing strategy, pig genetic makeup, and interaction with other vaccines. A total of 6,275 pigs were used to determine factors which affect circovirus vaccination and the effects of vaccination on average daily gain (ADG), immune responses, and viral circulation under field conditions. In the first study evaluating circovirus vaccination effects on PCV2 antibody titer, regardless of age and dose administration protocol, pigs vaccinated with a 2-dose circovirus vaccine had increased (P ≤ 0.008) antibody titers compared with non-vaccinates. In a second study, dosing strategy failed (P = 0.31) to affect antibody titers. However, product and time after vaccination did affect (P = 0.005) antibody titers. In another 130-d study across the nursery and finishing phases, pigs vaccinated with a 2-dose circovirus vaccine had decreased (P < 0.001) serum PCV2 viral load compared with non-vaccinates and ADG of vaccinates was better than non-vaccinates. However, the effect was more pronounced (vaccination-by-genetic interaction, P ≤ 0.05) in Duroc-based compared to Pietrain-based pigs. In a study limited to the nursery phase, vaccination for PCV2 and Mycoplasma hyopneumoniae independently reduced ADG and consumption, but the effect was product-dependent. In a 155-d study across the nursery and finishing phases, vaccination with a 2-dose, 2-vaccine program for PCV2 and Mycoplasma hyopneumoniae decreased (P < 0.001) nursery ADG but tended to increase (P = 0.06) finishing ADG compared to a 1-dose, 2-vaccine program, with no difference (P = 0.66) observed between final pig weights. Finally, circovirus vaccination affected PCV2-circulation in high-health research herds but not in a commercial herd where PCV2 DNA was detected in the environment. These results indicate that finishing performance was improved by a 2-dose circovirus vaccine; however, nursery performance was negatively affected by the same product. Circovirus vaccination responses of growth, viral load, and antibody titer were affected by pig genetic makeup, product, and PCV2-exposure status.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Joel M. DeRouchey / Steven S. Dritz / A total of 9,979 pigs were used in 11 experiments to quantify production responses under field conditions in growing pigs to PCV2 vaccination, biofuel co-products and dietary supplemental enzymes. Experiments 1 and 2 were conducted to determine the efficacy of a commercial 2-dose Porcine Circovirus Type 2 (PCV2) vaccine. Growth performance and mortality (P < 0.05) of vaccinated pigs improved compared to non-vaccinated pigs in both experiments with the vaccine causing a greater increase in ADG in vaccinated barrows than vaccinated gilts in Exp. 2. Experiment 3 compared the efficacy of 1-dose and 2-dose commercial PCV2 vaccines, where vaccinated pigs had greater ADG (P < 0.05) than vaccinated pigs regardless of vaccine type. The 2-dose group was heavier (P < 0.05) than the control group while the 1-dose group was intermediate. Therefore, PCV2 vaccines were efficacious under field conditions. Experiments 4, 5, and 6 were conducted to evaluate de-oiled corn dried distillers grains with solubles (dDGS) in grow-finish pigs. In Exp. 4, analyzed CP and AA content were higher, but lysine digestibility and energy content were lower in dDGS than traditional dried distillers grains with solubles (DDGS). In Exp. 5, 0 to 30% dDGS in nursery diets did not affect growth performance (P > 0.52). In Exp. 6, 0 to 30% dDGS reduced (linear; P < 0.01) ADG and ADFI, tended to improve (linear; P > 0.07) G:F, decreased (linear; P < 0.01) carcass yield, and increased (linear; P < 0.01) fat iodine values. Experiment 7 was conducted to determine the AA digestibility and energy concentration of novel high-CP distillers co-products from corn (HPC-DDG) and sorghum (HPS-DDGS). Digestibility of AA was higher for HPC-DDG but lower in HPS-DDGS than traditional DDGS. Both co-products had lower energy than traditional DDGS. Finally, Exp. 8, 9, 10, and 11 were used in a meta-analysis to evaluate supplementary dietary enzymes in pigs. Supplemental enzymes, alone or in combination, did not improve grow-finish pig performance (P > 0.58) regardless of dietary DDGS level. In conclusion, these experiments provide important empirical data to quantify production responses of various interventions and dietary ingredients under actual field conditions.

A suinocultura brasileira representa uma importante atividade econômica para o país, o qual ocupa lugar de destaque na produção e exportação de carne suína no mundo. O destaque do país na produção de suínos, deve-se a melhorias na sanidade, manejo, produção integrada e, principalmente, no aprimoramento gerencial dos produtores. O primeiro artigo consistiu em determinar a frequência e a distribuição das doenças infecciosas (DI) diagnosticadas através de exame de necropsia e histopatologia em suínos nas fases de crescimento e terminação ao longo de 12 anos (2005-2016) no sul do Brasil. Foram avaliados 1906 laudos anatomopatológicos de suínos nas fases de crescimento/terminação, dos quais as DI corresponderam a 75,6% (1441 casos) do total. As infecções por circovírus suíno tipo 2 (PCV2) foram as mais frequentes, contabilizando 51,3% (739/1441) dos casos, seguidas por DI que afetam o sistema respiratório (30,1% dos casos). Dentre essas, destacam-se a influenza suína A (15,1%; 218/1441) e pneumonias bacterianas (15%; 216/1441). O diagnóstico de influenza exibiu uma frequência elevada entre os anos de 2010 a 2013, totalizando 43,1% (167/387) dos casos. Após este período, ambas DI respiratórias exibiram caráter endêmico. As DI que afetam o sistema digestório totalizaram 10,5% (151/1441) dos diagnósticos, com as seguintes condições: enterocolite por Salmonella spp. (43,7%; 66/151), enteropatia proliferativa por Lawsonia spp. (41,7%; 63/151) e colite por Brachyspira spp. (14,6%; 22/151). Além dessas, as polisserosites e meningites bacterianas representaram 5,8% (84/1441) e 2,3% (33/1441) dos casos diagnosticados, respectivamente. O segundo artigo descreve três surtos de doença por circovírus suíno tipo-2 (PCVD) com lesões envolvendo musculatura esquelética. Em um curso clínico de 7 a 10 dias, 92 suínos apresentaram apatia, emagrecimento e diarreia. Ainda, cerca de 30 dos suínos afetados, apresentavam dificuldade de locomoção, fraqueza muscular, paresia de membros pélvicos e decúbito permanente. Quatro suínos exibiram palidez em músculos esqueléticos dos membros pélvicos, torácicos e dorso-lombares. As lesões microscópicas observadas consistiam de miosite necrótica granulomatosa, predominantemente, em membros pélvicos e torácicos, e em menor intensidade nos músculos dorso-lombares. No exame imuno-histoquímico para PCV2 observou-se marcação multifocal acentuada, predominantemente, no citoplasma e núcleos de macrófagos, linfócitos e células gigantes multinucleadas, além de marcação discreta no citoplasma no citoplasma de fibras necróticas da musculatura esquelética. As amostras de músculo esquelético foram positivas na reação em cadeia de polimerase para PCV2 e a ampliação exibiu 99% de identidade com sequências pertencentes ao genótipo PCV2b. / Brazilian pig farms represent an important economic activity for the country, which occupies a prominent place in the production and export of pork in the world. The country's prominence in pork production is due to improvements in sanitation, management, integrated production and, mainly, in the managerial improvement of the producers. The first study aimed to determine the frequency and distribution of infectious diseases (ID) diagnosed through necropsy and histopathology examination in growing-finishing swine along 12 years (2005-2016) in Southern Brazil. A total of 1906 anatomopathological exams performed in growing-finishing swine were evaluated, of which ID accounted for 75.6% (1441 cases) of the total. Porcine circovirus type 2 (PCV2) infections were the most frequent, accounting for 51.3% of the cases (739/1441), followed by respiratory system ID (30.1% of the cases). Among these, the main conditions were swine influenza (15.1%; 218/1441) and bacterial pneumonia (15%; 216/1441). Influenza diagnosis had a higher frequency between 2010 and 2013, accounting for 43,1% (167/387) of the cases. After this period, both respiratory ID had an endemic occurrence. Digestive system ID accounted for 10.5% (151/1441) of the diagnosis, with the main conditions diagnosed: Salmonella spp. enterocolitis (43.7%; 66/151), Lawsonia spp. proliferative enteropathy (41.7%; 63/151) and Brachyspira spp. colitis (14.6%; 22/151). Besides these, polyserositis and bacterial meningitis represented, respectively, 5.8% (84/1441) and 2.3% (33/1441) of the cases diagnosed. The second study describes three outbreak of porcine circovirus disease (PCVD) with the involvement of skeletal muscle. In a clinical course of 7 to 10 days, 92 pigs had apathy, weight loss and diarrhea. Approximately 30 of these 92 pigs had stiff gait, muscle weakness, hind limb paresis, and recumbency. 4 pigs necropsied presented pale discoloration from hind and thoracic limbs, as well from dorsal lumbar skeletal muscles. The microscopical lesions consisted of granulomatous necrotizing myositis mainly of hind and thoracic limbs, and mildly from dorsal lumbar muscles. Immunohistochemistry exam for PCV2 revealed marked multifocal intracytoplasmic and intranuclear staining predominantly in macrophages, lymphocytes and multinucleated giant cells, with a lower amount in the cytoplasm of necrotic fibers of the skeletal muscle. Affected muscle samples were polymerase chain reaction– positive for PCV2 and the amplicon exhibited 99% identity with sequences belonging to the PCV2b genotype.

Doctor of Philosophy / Diagnostic Medicine/Pathobiology / Raymond R. R. Rowland / Emerging viral diseases cause significant and widespread economic losses to U.S. swine production. Over the last 25 years, porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2) and porcine epidemic diarrhea virus (PEDV) have emerged or re-emerged, costing the industry billions through increased mortality and clinical or subclinical reductions in growth. Nursery pigs are greatly affected by these viruses due to high susceptibility to primary and secondary infections after weaning. However, clinical disease occurs in only a subpopulation of infected pigs and can vary drastically from sudden death to poor growth performance. This thesis documents a series of 4 studies where nursery pigs were challenged with either PRRSV/PCV2 or PEDV; the associations between clinical outcome and several factors affecting viral pathogenesis were investigated. In the first study, the administration of PRRS modified live virus vaccine prior to co-challenge with PRRSV/PCV2 was shown to protect against PRRS but enhance PCV2 replication and pathogenesis. This study provides insight into the role that PRRS vaccination has in both the control and potentiation of clinical disease. In the second study, microbial populations were compared between pigs with the best and worst clinical outcome following PRRSV/PCV2 co-infection. Increased fecal microbiome diversity was associated with improved clinical outcome; however, worst clinical outcome pigs had prolonged and greater virus replication, highlighting the host response to viral challenge as a primary determinant of clinical outcome. In the third study, 13 clinical phenotypes were compiled for >450 pigs after PRRSV/PCV2 co-infection. Duration of dyspnea and the presence of muscle wasting had the strongest associations with reduced weight gain. This study highlights the opportunity to improve animal welfare and production through improvements in clinical health. In the fourth study, clinical disease was mild to moderate and occurred within the first week after pigs were challenged with PEDV. However, PEDV was detected weeks after clinical disease had resolved and may implicate nursery pigs as an important source of viral carriage and transmission. Overall, the goal of this thesis was to develop models for understanding the impact of emerging and re-emerging viruses to improve recognition and control of disease.