Bovine herpesvirus-1 (BoHV-1) is a pathogen of cattle, which most commonly affects the upper respiratory tract to cause infectious bovine rhinotracheitis (IBR). It can also spread systemically to cause fatalities in calves and abortion in pregnant cattle. The virally encoded mechanisms of this systemic spread are poorly understood and therefore have been addressed by comparing isolates from the respiratory form of disease with isolates that have previously demonstrated systemic spread. A survey of 400 bovine abortions in Scotland from 2007-2009 demonstrated a BoHV-1 prevalence of 2.5%. It also demonstrated the importance of real-time PCR as a diagnostic technique when analysing samples from natural cases. The study of BoHV-1 distribution in the placenta and foetal tissue provided support for a haematogenous route of viral spread. Whole genome sequencing of 11 BoHV-1 isolates using Illumina Solexa technology was completed and added significantly to the sequencing data of BoHV-1. In terms of identifying genetic variation between isolates causing respiratory infection and those causing systemic infection, no differences were observed by SNP or phylogenetic analysis. However, there were significant differences in the extent of variation between essential and non-essential genes, which may reflect the evolution of BoHV-1. An in vivo challenge of the natural host to compare two isolates representing the respiratory and systemic forms of infection showed differences in clinical presentation, histopathological analysis, viral distribution and viral transcript expression, measured throughout the infection period. In particular, it was noted that a more severe ocular infection, rather than respiratory based infection was caused by infection with the ‘systemic’ isolate. Differences in the tropism of the virus were observed early in the infection with the ‘systemic’ isolate showing more association with the nasal mucosa than the trachea. The tonsils demonstrated different responses to the virus and differences in viral transcript expression. However, this may simply represent different stages of virus infection. Both isolates demonstrated spread to the brain at day 10 post infection. In vitro methods were used to study the differences in transcript expression in more detail. In a bovine turbinate cell infection faster replication of the respiratory isolate was observed by a significantly faster development of cytopathic effect. This was also reflected in the higher gene expression levels of the respiratory isolate in the first 12 hours of infection. More isolates were studied to investigate whether these differences were consistent, or as suggested by the sequencing, random differences between isolates. Six isolates were used to infect bovine lung slices. Differences in transcript expression were minimal between the two isolate groups. Immunofluorescence did not provide the sensitivity to detect virus in all samples where PCR showed replication. This compromised the study of co-localization but did show promise as a model to study the tropism of respiratory viruses. Overall, this work has showed that systemic spread of BoHV-1 does not appear to be controlled by virally encoded mechanisms. The in vivo experimental infection suggested host factors may play a large part. Further work is also needed to consider any differences that may exist between reactivated virus and the original infecting isolate.

Bovine herpesvirus-1 (BoHV-1) causes recurrent respiratory and genital infections in cattle; and predisposes them to lethal secondary bacterial infections. Vaccination is a primary strategy to prevent and reduce the severity of disease associated with BoHV-1, and to reduce virus transmission. While modified live (MLV) or killed (KV) BoHV-1 vaccines exist, these are expensive to produce, can cause disease (MLV) or may be ineffective (KV). Development of a DNA vaccine for BoHV-1 has the potential to address these shortcomings, but the very small amount of antigen expressed after DNA immunization presents a barrier to successful immunization of large animals. Engineering the vaccine to target this limited quantity of antigen to dendritic cells (DCs), the cells that prime immune responses, by attracting immature DCs (iDCs) to the vaccination site, is one way that DNA vaccine efficacy might be improved. Beta (β)-defensins are chemotactic peptides that, in studies with mice, improve induction of immune responses to DNA vaccines and this is due, at least in part, to their ability to attract iDCs to the site of vaccination. Accordingly, the objective of the studies described in this thesis was to determine whether using a bovine β-defensin in a DNA vaccine would enhance immune responses to the vaccine and subsequently protect cattle upon challenge with BoHV-1. First I characterized the bovine iDC and then used these cells to screen a panel of synthesized bovine β-defensins for chemotactic activity. The results showed that bovine neutrophil β-defensin (BNBD) 3, BNBD9 and enteric β-defensin (EBD) were equally the most chemotactic of the fourteen synthesized peptides for bovine iDCs. Because BNBD3 is the most abundant of the thirteen BNBDs and was able to attract CD1+ DCs when injected into the skin, I chose BNBD3 as the peptide I would use for the rest of the project. Next I constructed plasmids that expressed BNBD3; either alone or as a fusion construct with the BoHV-1 antigen truncated glycoprotein D (tgD), and then tested the effects of the plasmids as vaccines in both mice and cattle. In cattle, the addition of BNBD3 as a fusion strengthened the Th1 bias and increased cell-mediated immune responses to the DNA vaccine but not antibody response or protection from BoHV-1 infection. Given that inefficient humoral immune responses have been implicated in a lack of protection from BoHV-1 challenge, these results suggested that the successful BoHV-1 DNA vaccine would need to induce a much stronger humoral response. Lastly I assessed the ability of BNBD3 to improve humoral responses to pMASIA-tgD when complexed with the DNA vaccine and found that the vaccine complexed at a nanomolar peptide to DNA ratio of 125:1 increased humoral responses of mice. In vitro, treatment of mouse bone-marrow DCs with BNBD3 induced phenotypic and functional maturation/activation. This is an important aspect for vaccination in the skin, since after uptake, the DC must “mature” in order to traffic from the site of vaccination to the draining lymph node where induction of antigen-specific responses, by activated DCs, takes place. The findings in this thesis show that bovine β-defensins are chemotactic for bovine iDCs. I also show that using a bovine β-defensin as a fusion construct in a DNA vaccine enhances cell mediated but not humoral responses of cattle and yet this vaccine is protective against BoHV-1 challenge. I demonstrate that a bovine β-defensin, when used as a peptide to complex an antigen-encoding plasmid, can increase humoral responses. My work shows a multifunctional ability of bovine β-defensins to modulate and increase immune responses and suggests that bovine β-defensins likely have further untapped potential to enhance efficacy of DNA vaccines for large animals.

O complexo respiratório bovino é um dos principais problemas encontrados em bovinos tanto confinados quanto àqueles criados a pasto, levando a importantes perdas econômicas devido aos altos índices de morbidade e mortalidade. O objetivo geral deste trabalho foi determinar a prevalência de M. bovis, M. díspar, M. mycoides subsp. mycoides SC, P. multocida, M. haemolytica, Parainfluenza Bovino tipo-3 (PIb- 3) e Influenza vírus D e a prevalência de anticorpos contra Vírus da Diarreia Viral Bovina (VDVB), Herpesvírus tipo -1 (BoHV1) e o Vírus Respiratório Sincicial Bovino (VRSB) em bezerros sadios e com broncopneumonia criados nos assentamentos de Caiuá, Presidente Epitácio e Mirante de Paranapanema - SP. Além disso, objetivase avaliar a associação destes micro-organismos com a presença da broncopneumonia e dos sinais e sintomas apresentados pelos animais durante a avaliação física. Estudou-se 141 bezerros, machos e fêmeas de até um ano de idade, os quais, após exame físico, foram classificados em sadios e com pneumonia. Foram coletadas amostras de lavado traqueobrônquico e sangue total para obtenção do soro. As amostras foram utilizadas para isolamento e identificação de M. bovis, M. díspar, M. mycoides subsp. mycoides SC, P. multocida e M. haemolytica, detecção molecular de Parainfluenza Bovino tipo-3 (PIb-3) e Influenza vírus D (IVD) e detecção de anticorpos contra Vírus da Diarreia Viral Bovina (VDVB), Herpesvírus tipo -1 (HVBo-1) e o Vírus Respiratório Sincicial Bovino (VRSB). Não houve isolamento de P. multocida e M. haemolytica. Dentre as bactérias aeróbias isoladas, observou-se maior frequência de isolamento de Bacillus sp., Staphylococcus intermedius e bactérias Gram-negativas não fermentadoras. M. díspar foi a única espécie de micoplasma identificada com os oligonucleotídeos iniciadores utilizados. Faz-se necessário a busca por outras espécies de micoplasmas que podem estar relacionadas aos casos de broncopneumonia em bovinos. Não houve detecção de Parainfluenza Bovino tipo 3 e Influenza vírus D nas amostras de lavado traqueobrônquico. Os três municípios estudados apresentaram anticorpos para os vírus estudados. Observou-se prevalência de BoHV-1, VDVB e VRSB de 31,7%, 24,6 e 38,8%, respectivamente. Não houve associação entre o status de saúde dos bezerros e os achados microbiológicos e sorológicos. Foi observada associação entre enterobactérias e a variável “desidratação leve” (p=0,033/ OR= 11,25, IC: 1,809-41,834. Observou-se associações entre Mollicutes e a variável “secreção nasal serosa” (p=0,03). Concluiu-se que a broncopneumonia é uma enfermidade multifatorial. Faz-se, necessário, portanto, a associação dos achados de exames físico, microbiológicos e características do ambiente e manejo / Bovine respiratory disease complex is one of the major problems observed in feedlot and grazing cattle, causing economic losses due to high morbidity and mortality rates. The aim of this study was to determine the prevalence of M. bovis, M. díspar, M. mycoides subsp. mycoides SC, P. multocida, M. haemolytica, Bovine Parainfluenza type-3 (bPI-3) and Influenza vírus D (IVD) and the prevalence of antibodies against Bovine Viral Diarrhea Vírus (BVDV), Bovine Herpesvírus type -1 (BoHV1) and Bovine Respiratory Syncytial Vírus (VRSB) in healthy and pneumonic calves raised in settlements located in Caiuá, Presidente Epitácio e Mirante de Paranapanema São Paulo State. Moreover, we aimed to evaluate the association between those microorganisms and antibodies and the presence of bronchopneumonia and the symptoms observed during the physical examination. We studied 141 males and females calves that were classified as healthy and pneumonic calves. We collected tracheobronchial lavage samples and total blood to obtain serum samples. Isolation and biochemical/ molecular identification were performed in order to detect M. bovis, M. díspar, M. mycoides subsp. mycoides SC, P. multocida, M. haemolytica, Bovine Parainfluenza type-3 and Influenzavírus D. Serological survey was performed to detect antibodies aginst Bovine Viral Diarrhea Vírus (BVDV), Bovine Herpesvírus type -1 (BoHV1) and Bovine Respiratory Syncytial Vírus (VRSB). There was not any isolation of P. multocida and M. haemolytica. Bacillus sp., Staphylococcus intermedius and Non-fermenter Gram-negative bacteria were higher prevalent compared to the other aerobic bacteria isolated. M. díspar was the only mycoplasma species identified by the primers used. It is necessary the identification of other mycoplasma species that could be related to the cases of bronchopneumonia in cattle. Bovine Parainfluenza type-3 and Influenza vírus D were not identified. Antibodies were detected in all municipalities. The prevalence of BoHV, BVDV e VRSB was 31.7%, 24.6 and 38.8%, respectively. The association between health status and microbial/ serological findings was not observed. Enterobacterias were associated to “mild dehydration” (p=0.033/OR=11.25; IC: 1.809-41.834). Associations between Mollicutes and “serous nasal discharge” (p=0.03) was also detected. In conclusion, S. intermedius was associated to “nasal air flow” (p=0.097). In conclusion bovine bronchopneumonia is multi-factorial disease. It is necessary, though, physical examination, microbiology and management and environment feature results

A agropecuária familiar brasileira produz 70% da alimentação da população, possuindo importante papel social e econômico neste país. Os assentamentos do Pontal do Paranapanema produzem alimentos que são ofertados ao estado de São Paulo, sendo atividade leiteira a mais importante. As doenças infecciosas reprodutivas dos bovinos, causados por Leptospira spp., Herpes Virus Bovino Tipo 1(BoHV-1), Diarreia Viral Bovina (BVD), agentes da classe Mollicutes são importantes para a rentabilidade das produções agropecuárias do mundo inteiro. Dados epidemiológicos nos assentamentos do estado de São Paulo não existem. Este estudo teve como objetivo analisar a prevalência das enfermidades reprodutivas bacterianas e virais em bovinos de leite criados nos assentamento de Presidente Epitácio e Mirante do Paranapanema. Todos os animais foram submetidos ao exame específico do sistema reprodutivo e colheram-se amostras de soro e swab vaginal. A prevalência para BoHV-1, BVDV, Leptospira spp., e classe Mollicutes foi de 69,05%, 57,74%, 58,93% e 50%, respectivamente. Foi observado uma relação entre vacas multíparas e a presença de BoHV-1 e Leptospira spp. Foi observado também uma relação de animais com retenção de placenta com a presença de bactérias da classe Mollicutes e animais reagentes a anticorpos contra Leptospira spp. A prevalência destas enfermidades prova a existência destas em assentamentos na região do Pontal do Paranapanema, faz-se necessário o desenvolvimento de programas de políticas públicas para o monitoramento desta região / Brazilian\'s family settlements are responsible for 70% of total population feeding. It has an important social and economic role in Brazil. Settlements from Pontal do Paranapanema offering feed to the state of São Paulo and milk production is the most important activity. Bovine reproduction infectious diseases caused by Leptospira spp., Bovine Herpesvirus Type 1(BoHV-1), Bovine viral Diarrhoea (BVD) and Mollicutes are important to the dairy cattle industry all over the world. Epidemiological datas from settlements located in the state of São Paulo are not available. The aim of this study was to evaluate the prevalence of bacterial and viral reproductive infectious diseases dairy cattle raised in settlements from Presidente Epitácio e Mirante do Paranapanema. Specific physical examination of reproduction female system was performed and after that vaginal swabs samples and serum samples were collected. The prevalence of BoHV-1, BVDV, Leptospira spp., and Mollicutes was 69,05%, 57,74%, 58,93%, and 50%, respectively. An association between multiparous cows and the presence of BoHV-1 and Leptospira spp., was detected. Also an association between females with retained placenta and Mollicutes and cows with antibodies against Leptospira spp. was observed. The results showed the presence of reproduction diseases in settlements in Pontal do Paranapanema and it is necessary the development of public politics programs to control them

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5) are genetic and antigenic closely related pathogens of cattle, historically associated with respiratory/genital and neurological disease, respectively. This thesis reports the genomic and phylogenetic analyzes based on glycoproteins C (gC) and D (gD) genes of BoHV-1 and BoHV-5. Initially, the differentiation between BoHV-1 and BoHV-5 was performed by a differential PCR based on 5 gC gene. Then, a molecular analysis based on the 3 region of the gC gene of 45 BoHV isolates from Brazil, Uruguay and Argentina (1981-2009) was described. The phylogenetic tree reconstruction provided a clear distinction between BoHV-1 and BoHV-5, and BoHV-1 into subtypes BoHV-1.1 and BoHV-1.2. The levels of nucleotide (nt) similarity ranged from 99.1 to 100% among BoHV-1 sequences (n=12); 96.2-100% among BoHV-5 sequences (n=32); and 77.7-90.3% between BoHV-1 and BoHV-5 sequences. A transmembrane domain of 24 amino-acid (aa) and the putative cytoplasmic tail of 8 aa were also identified. In addition, a phylogenetic study was performed to investigate genetic divergences at the 3 region of gD gene of respiratory/genital BoHV-1 (n=7), neurological BoHV-1 (n=7) and neurological BoHV-5 (n=7) isolates, and whether these differences would be associated with the respective neurological presentation. The phylogenetic reconstruction allowed a clear differentiation of BoHV-1 (n=14) and BoHV-5 (n=7), but BoHV-1 isolates from neurological disease grouped within BoHV-1 branch. The nt and aa similarity levels were on average 98.3% among BoHV-1; 97.8% and 95.8% among BoHV-5; 73.7% and 64.1% between viral species. These results indicate that both genes revealed a high conserved 3 region within each species and a less conserved region between BoHV-1 and BoHV-5. The phylogenetic analyzes allowed differentiation of BoHV-1 and BoHV-5 species, and even subtypes grouped in distinct branches in the 3 gC gene-based study, indicating that this region represents a better choice for phylogenetic subgrouping. So, it was concluded that these genome regions represent a suitable target for phylogenetic classification of BoHV-1 and BoHV-5 isolates, and, perhaps, for understanding evolutionary relationships. However, no conclusion of a possible association of genetic differences with phenotypes could be drawn. / Bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5) are genetic and antigenic closely related pathogens of cattle, historically associated with respiratory/genital and neurological disease, respectively. This thesis reports the genomic and phylogenetic analyzes based on glycoproteins C (gC) and D (gD) genes of BoHV-1 and BoHV-5. Initially, the differentiation between BoHV-1 and BoHV-5 was performed by a differential PCR based on 5 gC gene. Then, a molecular analysis based on the 3 region of the gC gene of 45 BoHV isolates from Brazil, Uruguay and Argentina (1981-2009) was described. The phylogenetic tree reconstruction provided a clear distinction between BoHV-1 and BoHV-5, and BoHV-1 into subtypes BoHV-1.1 and BoHV-1.2. The levels of nucleotide (nt) similarity ranged from 99.1 to 100% among BoHV-1 sequences (n=12); 96.2-100% among BoHV-5 sequences (n=32); and 77.7-90.3% between BoHV-1 and BoHV-5 sequences. A transmembrane domain of 24 amino-acid (aa) and the putative cytoplasmic tail of 8 aa were also identified. In addition, a phylogenetic study was performed to investigate genetic divergences at the 3 region of gD gene of respiratory/genital BoHV-1 (n=7), neurological BoHV-1 (n=7) and neurological BoHV-5 (n=7) isolates, and whether these differences would be associated with the respective neurological presentation. The phylogenetic reconstruction allowed a clear differentiation of BoHV-1 (n=14) and BoHV-5 (n=7), but BoHV-1 isolates from neurological disease grouped within BoHV-1 branch. The nt and aa similarity levels were on average 98.3% among BoHV-1; 97.8% and 95.8% among BoHV-5; 73.7% and 64.1% between viral species. These results indicate that both genes revealed a high conserved 3 region within each species and a less conserved region between BoHV-1 and BoHV-5. The phylogenetic analyzes allowed differentiation of BoHV-1 and BoHV-5 species, and even subtypes grouped in distinct branches in the 3 gC gene-based study, indicating that this region represents a better choice for phylogenetic subgrouping. So, it was concluded that these genome regions represent a suitable target for phylogenetic classification of BoHV-1 and BoHV-5 isolates, and, perhaps, for understanding evolutionary relationships. However, no conclusion of a possible association of genetic differences with phenotypes could be drawn.

Bovine herpesvirus type 1 (BoHV-1) is an important pathogen of cattle and causes significant economic losses to livestock industry. Monoclonal antibodies (mAbs) represent useful tools for diagnostic and research purposes. Most mAbs produced against BoHV-1 are directed to glycoprotein gC (gC), an abundant and immunodominant envelope antigen. In the present study, antigens of a BoHV-1 strain defective in the gene coding for glycoprotein C was used to immunize BALB/c mice to produce mAbs with other protein specificities. After fusion and selection of 54 hybridomas resistant to the selective medium HAT, three hybridomas secreting mAbs directed to BoHV-1 antigens were obtained (1F1, 2H4, 4D7). The mAbs belong to the IgG2a isotype and reacted in an indirect fluorescent antibody assay (IFA) and indirect immunoperoxidase staining (IPX) of BoHV-1-infected cells at dilutions up to 1:640 (culture supernatant) and 1:20,000 (ascitic fluid). The three mAbs tested reacted with 14 isolates of respiratory and/or genital disease and with 17 isolates of neurological disease and showed variable level of neutralizing activity against the most of these isolates. The protein specificity of the mAbs could not be determined, because none of them reacted with viral proteins in western immunoblot. On the other hand, the three mAbs reacted in IFA with viral antigens of BoHV-1 and BoHV-5 mutant strains defective on gC, gE, gI and US9 genes, demonstrating that they are directed against other viral antigens. Because the high reaction titer and the wide range of reactivity, these mAbs have potential use in diagnostics techniques. Also, these mAbs may be useful to map conserved neutralizing epitopes in the envelope glycoproteins / O herpesvírus bovino tipo 1 (BoHV-1) é um dos principais patógenos de bovinos e causa perdas significativas para a bovinocultura. Anticorpos monoclonais (AcMs) se constituem em importantes ferramentas para o diagnóstico e pesquisa em diversos aspectos da biologia desse agente. A maioria dos AcMs produzidos contra o BoHV-1 são direcionados contra a glicoproteína C (gC), um antígeno abundante e imunodominante do envelope viral. No presente trabalho, antígenos de uma cepa do BoHV-1 defectiva no gene da gC foram utilizados para a imunização de camundongos visando a produção de AcMs com outras especificidades protéicas. Após fusão e seleção de 54 clones resistentes ao meio seletivo HAT, foram obtidos três hibridomas secretores de AcMs contra antígenos do BoHV-1 (1F1, 2H4, 4D7). Os AcMs pertenceram ao isotipo IgG2a e reagiram em diluições de até 1:640 (sobrenadante de cultivo) e 1:20.000 (fluído ascítico) em testes de imunofluorescência indireta (IFA) e imunoperoxidase indireta (IPX). Os três AcMs reagiram na IFA com 14 isolados de doença respiratória e/ou genital e com 17 isolados de doença neurológica, e apresentaram atividade neutralizante em níveis variáveis contra a grande maioria desses isolados. A especificidade protéica dos AcMs não foi determinada, pois nenhum deles reagiu com proteínas virais na técnica de Western blot. Por outro lado, os três AcMs reagiram na IFA contra antígenos virais de cepas do BoHV-1 e BoHV-5 com deleção dos genes que codificam as proteínas gC, gE, gI e US9, demonstrando que são direcionados contra outros antígenos virais. Pelo seu alto título de reação e pelo amplo espectro de reatividade, esses AcMs possuem aplicação potencial em técnicas diagnósticas. Esses AcMs também podem ser úteis para o mapeamento de epitopos neutralizantes conservados nas glicoproteínas do envelope

Made available in DSpace on 2015-03-26T13:47:03Z (GMT). No. of bitstreams: 1 texto completo.pdf: 637342 bytes, checksum: 6a735b2fcb2171581a05241eb10ef371 (MD5) Previous issue date: 2012-02-10 / In order to clarify data on the dynamics of infection by Bovine herpesvirus 1 (BoHV-1) and / or Bovine herpesvirus 5 (BoHV-5), the aim of this study was to compare the viral clearance of BoHV-1 and/or BoHV-5 in two distinct phases of lactation (five months before and at parturition) of naturally infected dairy cows. Measurements of the cortisol hormone were used as an analysis parameter in order to relate viral shedding to post partum stress., The elimination of viral DNA in colostrum / milk, nasal secretions and viremia was investigated by nested PCR. The antibody response of cattle naturally infected was investigated by neutralization. There was no difference in cortisol levels between the two lactation phases. Therefore, there was no difference in viral genome detection and antibody responses of the tested samples, considering the two studied phases. Milk was an effective sample alternative for genomic diagnosis of BoHV-1 and/or BoHV-5. In one of the properties studied, close to 100% of infected animals were shedding viral DNA, and the mean frequency of milk shedding in cows from other properties was only 52%. The positive viremia rate was similar to the one observed in the milk of infected animals, while viral clearance in nasal discharges had low positivity rate, compared to milk. This suggests an extended shedding time in the milk, when compared to other dissemination routes. This information corroborates with milk efficacy as a sample alternative for viral diagnosis. Thus, in this study, Nested- PCR is an efficient tool for the viral diagnosis in milk. Milk is a promising sample for field practitioners because not only does it allow for productive infection diagnosis, it is easier to collect compared to blood. / A fim de esclarecer dados da dinâmica da infecção pelos Bovine herpesvirus 1 (BoHV-1) e/ou Bovine herpesvirus 5 (BoHV-5), este trabalho visou comparar a eliminação dos referidos agentes virais em duas fases distintas da lactação (cinco meses antes e na época do parto) de vacas leiteiras naturalmente infectadas. Isso, de modo a relacionar a eliminação viral ao estresse periparto, tomando como parâmetro de análise dosagens do hormônio cortisol. Também foi investigada, através da Nested-PCR, a eliminação do DNA viral no colostro/leite, secreção nasal, viremia e, através da soroneutralização, a resposta sorológica de vacas leiteiras naturalmente infectadas. Foi demonstrado não haver diferenças dos níveis de cortisol entre as fases produtivas estudadas. Consequentemente, não houve diferenças na detecção do genoma viral e na resposta humoral das amostras clínicas focadas, considerando as duas fases estudadas. O leite se apresentou como uma alternativa amostral eficaz de diagnóstico da presença genômica do BoHV-1 e/ou BoHV-5. Em uma das propriedades de trabalho, praticamente 100% dos animais infectados estavam eliminando material genômico dos agentes, e houve uma média de 52% de frequência de positividade desse material eliminado no leite das vacas das demais propriedades. A frequência positiva de viremia se assemelhou àquela do leite em animais infectados, enquanto que a eliminação via secreção nasal apresentou baixa frequência de positividade, comparando ao leite. Isso sugere um prolongamento da época de eliminação via leite quando comparada à secreção nasal, concordando com a eficácia do leite como alternativa de material para diagnóstico viral. Sendo assim, conclui-se que a Nested-PCR é um método eficiente para o diagnóstico dos vírus deste estudo em leite e que essa amostra é promissora para o foco dos profissionais de campo, pois, além do diagnóstico ser possível na fase de infecção produtiva, há maior facilidade em sua coleta, quando comparada à coleta de sangue.

O complexo respiratório bovino é um dos principais problemas encontrados em bovinos tanto confinados quanto àqueles criados a pasto, levando a importantes perdas econômicas devido aos altos índices de morbidade e mortalidade. O objetivo geral deste trabalho foi determinar a prevalência de M. bovis, M. díspar, M. mycoides subsp. mycoides SC, P. multocida, M. haemolytica, Parainfluenza Bovino tipo-3 (PIb- 3) e Influenza vírus D e a prevalência de anticorpos contra Vírus da Diarreia Viral Bovina (VDVB), Herpesvírus tipo -1 (BoHV1) e o Vírus Respiratório Sincicial Bovino (VRSB) em bezerros sadios e com broncopneumonia criados nos assentamentos de Caiuá, Presidente Epitácio e Mirante de Paranapanema - SP. Além disso, objetivase avaliar a associação destes micro-organismos com a presença da broncopneumonia e dos sinais e sintomas apresentados pelos animais durante a avaliação física. Estudou-se 141 bezerros, machos e fêmeas de até um ano de idade, os quais, após exame físico, foram classificados em sadios e com pneumonia. Foram coletadas amostras de lavado traqueobrônquico e sangue total para obtenção do soro. As amostras foram utilizadas para isolamento e identificação de M. bovis, M. díspar, M. mycoides subsp. mycoides SC, P. multocida e M. haemolytica, detecção molecular de Parainfluenza Bovino tipo-3 (PIb-3) e Influenza vírus D (IVD) e detecção de anticorpos contra Vírus da Diarreia Viral Bovina (VDVB), Herpesvírus tipo -1 (HVBo-1) e o Vírus Respiratório Sincicial Bovino (VRSB). Não houve isolamento de P. multocida e M. haemolytica. Dentre as bactérias aeróbias isoladas, observou-se maior frequência de isolamento de Bacillus sp., Staphylococcus intermedius e bactérias Gram-negativas não fermentadoras. M. díspar foi a única espécie de micoplasma identificada com os oligonucleotídeos iniciadores utilizados. Faz-se necessário a busca por outras espécies de micoplasmas que podem estar relacionadas aos casos de broncopneumonia em bovinos. Não houve detecção de Parainfluenza Bovino tipo 3 e Influenza vírus D nas amostras de lavado traqueobrônquico. Os três municípios estudados apresentaram anticorpos para os vírus estudados. Observou-se prevalência de BoHV-1, VDVB e VRSB de 31,7%, 24,6 e 38,8%, respectivamente. Não houve associação entre o status de saúde dos bezerros e os achados microbiológicos e sorológicos. Foi observada associação entre enterobactérias e a variável “desidratação leve” (p=0,033/ OR= 11,25, IC: 1,809-41,834. Observou-se associações entre Mollicutes e a variável “secreção nasal serosa” (p=0,03). Concluiu-se que a broncopneumonia é uma enfermidade multifatorial. Faz-se, necessário, portanto, a associação dos achados de exames físico, microbiológicos e características do ambiente e manejo / Bovine respiratory disease complex is one of the major problems observed in feedlot and grazing cattle, causing economic losses due to high morbidity and mortality rates. The aim of this study was to determine the prevalence of M. bovis, M. díspar, M. mycoides subsp. mycoides SC, P. multocida, M. haemolytica, Bovine Parainfluenza type-3 (bPI-3) and Influenza vírus D (IVD) and the prevalence of antibodies against Bovine Viral Diarrhea Vírus (BVDV), Bovine Herpesvírus type -1 (BoHV1) and Bovine Respiratory Syncytial Vírus (VRSB) in healthy and pneumonic calves raised in settlements located in Caiuá, Presidente Epitácio e Mirante de Paranapanema São Paulo State. Moreover, we aimed to evaluate the association between those microorganisms and antibodies and the presence of bronchopneumonia and the symptoms observed during the physical examination. We studied 141 males and females calves that were classified as healthy and pneumonic calves. We collected tracheobronchial lavage samples and total blood to obtain serum samples. Isolation and biochemical/ molecular identification were performed in order to detect M. bovis, M. díspar, M. mycoides subsp. mycoides SC, P. multocida, M. haemolytica, Bovine Parainfluenza type-3 and Influenzavírus D. Serological survey was performed to detect antibodies aginst Bovine Viral Diarrhea Vírus (BVDV), Bovine Herpesvírus type -1 (BoHV1) and Bovine Respiratory Syncytial Vírus (VRSB). There was not any isolation of P. multocida and M. haemolytica. Bacillus sp., Staphylococcus intermedius and Non-fermenter Gram-negative bacteria were higher prevalent compared to the other aerobic bacteria isolated. M. díspar was the only mycoplasma species identified by the primers used. It is necessary the identification of other mycoplasma species that could be related to the cases of bronchopneumonia in cattle. Bovine Parainfluenza type-3 and Influenza vírus D were not identified. Antibodies were detected in all municipalities. The prevalence of BoHV, BVDV e VRSB was 31.7%, 24.6 and 38.8%, respectively. The association between health status and microbial/ serological findings was not observed. Enterobacterias were associated to “mild dehydration” (p=0.033/OR=11.25; IC: 1.809-41.834). Associations between Mollicutes and “serous nasal discharge” (p=0.03) was also detected. In conclusion, S. intermedius was associated to “nasal air flow” (p=0.097). In conclusion bovine bronchopneumonia is multi-factorial disease. It is necessary, though, physical examination, microbiology and management and environment feature results

A agropecuária familiar brasileira produz 70% da alimentação da população, possuindo importante papel social e econômico neste país. Os assentamentos do Pontal do Paranapanema produzem alimentos que são ofertados ao estado de São Paulo, sendo atividade leiteira a mais importante. As doenças infecciosas reprodutivas dos bovinos, causados por Leptospira spp., Herpes Virus Bovino Tipo 1(BoHV-1), Diarreia Viral Bovina (BVD), agentes da classe Mollicutes são importantes para a rentabilidade das produções agropecuárias do mundo inteiro. Dados epidemiológicos nos assentamentos do estado de São Paulo não existem. Este estudo teve como objetivo analisar a prevalência das enfermidades reprodutivas bacterianas e virais em bovinos de leite criados nos assentamento de Presidente Epitácio e Mirante do Paranapanema. Todos os animais foram submetidos ao exame específico do sistema reprodutivo e colheram-se amostras de soro e swab vaginal. A prevalência para BoHV-1, BVDV, Leptospira spp., e classe Mollicutes foi de 69,05%, 57,74%, 58,93% e 50%, respectivamente. Foi observado uma relação entre vacas multíparas e a presença de BoHV-1 e Leptospira spp. Foi observado também uma relação de animais com retenção de placenta com a presença de bactérias da classe Mollicutes e animais reagentes a anticorpos contra Leptospira spp. A prevalência destas enfermidades prova a existência destas em assentamentos na região do Pontal do Paranapanema, faz-se necessário o desenvolvimento de programas de políticas públicas para o monitoramento desta região / Brazilian\'s family settlements are responsible for 70% of total population feeding. It has an important social and economic role in Brazil. Settlements from Pontal do Paranapanema offering feed to the state of São Paulo and milk production is the most important activity. Bovine reproduction infectious diseases caused by Leptospira spp., Bovine Herpesvirus Type 1(BoHV-1), Bovine viral Diarrhoea (BVD) and Mollicutes are important to the dairy cattle industry all over the world. Epidemiological datas from settlements located in the state of São Paulo are not available. The aim of this study was to evaluate the prevalence of bacterial and viral reproductive infectious diseases dairy cattle raised in settlements from Presidente Epitácio e Mirante do Paranapanema. Specific physical examination of reproduction female system was performed and after that vaginal swabs samples and serum samples were collected. The prevalence of BoHV-1, BVDV, Leptospira spp., and Mollicutes was 69,05%, 57,74%, 58,93%, and 50%, respectively. An association between multiparous cows and the presence of BoHV-1 and Leptospira spp., was detected. Also an association between females with retained placenta and Mollicutes and cows with antibodies against Leptospira spp. was observed. The results showed the presence of reproduction diseases in settlements in Pontal do Paranapanema and it is necessary the development of public politics programs to control them

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No. of bitstreams: 2 Dissertação - Amanda Vargas Teles - 2016.pdf: 2056252 bytes, checksum: 368e99b26e688ce89da00fe5b604b179 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2016-02-29 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Bovine herpesvirus 1 (BoHV-1) is a virus of the family Herpesviridae and the subfamily Alphaherpesvirinae, known to cause several diseases worldwide in cattle, including rhinotracheitis, vaginitis, balanoposthitis, abortion, conjunctivitis, and enteritis. O BoHV-1 is a source of economic loss for both dairy and beef producer due to a decrease in dairy production, decrease in animal weight gain, a higher susceptibility to secondary infections, and the occurrences of abortions. Aiming at new alternatives for the treatment and disposal of these cattle pathogen, the photoinactivation becomes a viable alternative because this treatment has shown to be effective against animal and human microorganisms. The technique is based on the synergistic combination of a photosensitizer (PS), oxygen, and light. When irradiated by a light source, the photosensitizer undergoes a series of photophysical processes, which culminate in the generation of reactive oxygen species (ROS), which in large amounts are toxic to pathogens. Aiming at a better efficiency of photoinactivation is necessary to know the characteristics of each photosensitizer, analyzing the molecular structure, quantum yield, lifetime, toxicity, stability, and absorption at specific wavelengths. The irradiation time has shown to be very important because each PS reacts differently at a given time. To evaluate the effect of the electrical charges present on PS, zinc insert ring in the central ring of the molecule and the irradiation time in the photodynamic inactivation, BoHV-1 aliquots were used with initial viral titer from 105.75 incubated for one hour with four different photosensitizers: TPPS4, ZnTPPS4, TMPyP, and ZnTMPYP. Subsequently, they we irradiated for two hours and sampled every 15 minutes. As control, we used virus without any treatment, virus irradiated in the absence of PS, and virus with PS without radiating. After irradiation, the sample was added to cultures of cells permissive to the virus, which was analyzed for the presence of cytopathic effect (CPE), and the results were expressed as viral titers. Although all treatments had significant differences, all PSs used in the study were effective in the treatment of BoHV-1. The ZnTMPYP was the most efficient, fully inactivating the virus with 30 minutes of irradiation, and the TPPS4 showed the worst result unable to inactivate the virus after the period of irradiation, but considerably reduced the viral load of BoHV -1. We also analyzed the photodegradation of a plasmid DNA using the same photosensitizers. Again, ZnTMPYP showed better results. / O herpesvírus bovino 1 (BoHV - 1) é um vírus da família Herpesviridae e da subfamília Alphaherpesvirinae, conhecido por causar diversas doenças em bovinos no mundo todo, incluindo a rinotraqueíte, vaginite, balanopostite, aborto, conjuntivite e enterite. O BoHV - 1 é responsável por grande perda econômica, tanto para produtores de leite quanto de carne, devido a uma diminuição na produção de leite, diminuição na taxa de ganho de peso animal, uma maior susceptibilidade às infecções secundárias e as ocorrências de abortos. Visando novas alternativas para o tratamento e eliminação deste patógeno do rebanho, a fotoinativação se torna uma alternativa viável devido ao fato deste tratamento ter se mostrado eficiente contra vários microrganismos de interesse animal e humano. A inativação fotodinâmica (PDI) é baseada na combinação de três componentes: fotossensibilizador (PS), oxigênio e luz. Ao ser irradiado por uma fonte de luz no comprimento de onda específico, o fotossensibilizador sofre uma série de processos fotofísicos que culminarão na geração de espécies reativas do oxigênio (ROS) que em grandes quantidades são tóxicos a patógenos. Visando uma melhor eficiência da fotoinativação, é necessário conhecer as características de cada fotossensibilizador, analisando a estrutura molecular, rendimento quântico, tempo de vida, toxicidade, estabilidade e absorção em comprimentos de onda específicos. O tempo de irradiação tem se mostrado muito importante, porque cada PS reage de uma maneira diferente em determinado período. Para avaliar a interferência das cargas elétricas presentes nos PS, a inserção de zinco no anel no anel central da molécula e o tempo de irradiação na inativação fotodinâmica, foram utilizadas alíquotas do BoHV-1 com título viral inicial de 105,75, incubadas durante uma hora com quatro diferentes porfirinas como fotossensibilizadores: TPPS4, ZnTPPS4, TMPYP e ZnTMPYP. Posteriormente, foram irradiadas durante duas horas, sendo retiradas amostras a cada 15 minutos. Como controle foram utilizados vírus sem qualquer tipo de tratamento, vírus irradiado sem a presença de PS e vírus com PS sem irradiar. Após a irradiação a amostra foi adicionada em culturas de células permissíveis ao vírus, onde foi analisada a presença de efeito citopático (CPE) e os resultados foram expressos em títulos virais. Apesar de todos os tratamentos possuírem diferenças significativas, todos os PSs utilizados no estudo se mostraram eficazes no tratamento do BoHV-1. A ZnTMPYP foi a mais eficiente, inativando totalmente o vírus com 30 minutos de irradiação, e a TPPS4 apresentou o pior resultado não conseguindo inativar o vírus após o período de irradiação, porém reduziu consideravelmente a carga viral do BoHV-1. Analisamos também a fotodegradação de um DNA plasmidial utilizando os mesmos fotossensibilizadores. Novamente, a ZnTMPYP apresentou melhores resultados.