Infectious disease is an important issue for animal breeders, farmers and governments. Solutions to control infectious disease are needed and research focused on the genetic loci determining variation in immune-related traits has the potential to deliver solutions. The primary aim of this thesis is to discover regions of the bovine genome which influence the immune response post immunisation. To accomplish this two types of immunising agents, a Foot-and-Mouth Disease Virus (FMDV) peptide (FMDV15) and a commercial vaccine for Bovine Respiratory Syncytial Virus (BRSV), were used to immunise the second generation (F2 and backcrosses) of the Roslin Bovine Genome (RoBoGen) herd, a Charolais Holstein cross population. The FMDV15 peptide consisted of two sections of the VP1 protein located on the FMDV capsid, together encompassing the major neutralising antibody sites that are known to be immunogenic. Protection against FMDV is generally believed to relate to the levels of neutralising antibody and has been correlated with IgG1 and IgG2 levels as well as interferon- . In addition it has been shown that T cell responses also play a role in protection against FMDV. Thus all of these were used as phenotypic measurements post immunisation to the FMDV15 peptide. The BRSV vaccine used was an attenuated live vaccine. Protective mechanisms against BRSV infection include IgA, IgG1, IgG2 and IgM BRSV-specific antibodies and antibody titres particularly those of the IgG isotypes are considered to be correlates of protection. Thus, IgG1 and IgG2 antibody levels were measured post vaccination with the BRSV vaccine. All phenotypes were measured across time, and allowed analysis of the primary and secondary adaptive immune responses. Both agents caused considerable variation in the phenotypes measured post immunisation, with significant responses detected two weeks post immunisation. REstricted Maximum Likelihood (REML) analysis attributed much of this variation to sire, highlighting the heritable component, and environmental effects. Significant positive correlations were detected across time within each trait for both the FMDV and BRSV responses. The FMDV and BRSV antibody levels also correlated with each other at later time points, suggesting that there may be animals which are genetically predisposed to be high or low responders in general. Initially a linkage mappingapproach was followed using 165 microsatellite markers, which detected 77 QTL in response to the FMDV peptide and 27 QTL in response to the BRSV vaccine. There were some overlapping QTL, for example QTL which spanned the Major Histocompatibility Complex. Further analysis was conducted by developing a Perl scripted program which genotyped the RoBoGen herd in two ways; 1) Single Nucleotide Polymorphism(s) (SNP) were genotyped within the confidence intervals of the previously discovered QTL and 2) SNP were genotyped via a candidate gene approach. Association study methodology, accounting for relationship stratification via principal components of the genetic relationship matrix, was used to detect significant SNP, in response to both the FMDV peptide and the BRSV vaccine. Twenty significant SNP associations were discovered across 19 traits, with some SNP located in genes with known biological relevance to an immune response, such as the Toll-Like Receptors (TLR), TLR4 and TLR8. This thesis has detected regions of the genome which are significantly associated with the immune responses elicited by two different agents, suggesting similar pathway(s)/gene(s) may be used in defence of multiple pathogens. Once regions of significance were detected, further analysis using SNP markers identified significant, non-synonymous SNP that were associated with the immunising agents. The novel markers discovered in this study may aid breeding for resistance to disease via marker assisted selection. In addition, they may also have highlighted new targets for vaccinologists to develop ‘next generation’ vaccines.

As respiratory syncytial virus (RSV) is a respiratory pathogen that causes significant morbidity and mortality in infants, there has always been great interest in the development of a vaccine. In the 1960s, children were immunized with formalin-inactivated (FI)-RSV vaccines. Not only did these vaccines fail to prevent infection, but in most cases they resulted in enhanced disease upon subsequent exposure to the virus. In the intervening years, studies in mice have led to the hypothesis that the enhanced disease is due to an aberrant Th2-biased immune response. Thus, we hypothesized that formulating FI-RSV vaccines with a Th1 promoting adjuvant, such as CpG oligoeoxynucleotides (ODN), would result in the induction of protective immunity against RSV without risk of deleterious effects. We observed in calves that parenterally delivered FI-bovine RSV (BRSV) formulated with CpG ODN resulted in a shift towards a Th1-biased or more balanced immune response that was protective against BRSV.<p> As RSV infects the lung mucosa, vaccines that induce mucosal immunity are desirable. Parenterally delivered vaccines typically induce systemic immunity with low mucosal immune response levels, whereas mucosally delivered vaccines induce systemic and mucosal immunity. However, upon mucosal delivery there is an increased chance of vaccine components being degraded or washed away prior to the induction of immunity. Thus, we added polyphosphazenes (PP) to our mucosal vaccine formulations. PP are synthetic polymers that form non-covalent complexes with other vaccine components, increasing their stability. Intranasally delivered FI-BRSV co-formulated with CpG ODN and PP performed better than FI-BRSV alone, or FI-BRSV formulated with either adjuvant individually, in terms of inducing protective immunity against BRSV in mice. Furthermore, mice that received intranasally-delivered FI-BRSV or BRSV F protein co-formulated with CpG ODN and PP developed higher levels of immunity and protection than mice that received parenterally delivered vaccines. Because of the similarities between BRSV and HRSV, co-formulation of intranasally delivered HRSV vaccines with CpG ODN and PP could prove important in the development of a safe vaccine against HRSV in humans.

As respiratory syncytial virus (RSV) is a respiratory pathogen that causes significant morbidity and mortality in infants, there has always been great interest in the development of a vaccine. In the 1960s, children were immunized with formalin-inactivated (FI)-RSV vaccines. Not only did these vaccines fail to prevent infection, but in most cases they resulted in enhanced disease upon subsequent exposure to the virus. In the intervening years, studies in mice have led to the hypothesis that the enhanced disease is due to an aberrant Th2-biased immune response. Thus, we hypothesized that formulating FI-RSV vaccines with a Th1 promoting adjuvant, such as CpG oligoeoxynucleotides (ODN), would result in the induction of protective immunity against RSV without risk of deleterious effects. We observed in calves that parenterally delivered FI-bovine RSV (BRSV) formulated with CpG ODN resulted in a shift towards a Th1-biased or more balanced immune response that was protective against BRSV.<p> As RSV infects the lung mucosa, vaccines that induce mucosal immunity are desirable. Parenterally delivered vaccines typically induce systemic immunity with low mucosal immune response levels, whereas mucosally delivered vaccines induce systemic and mucosal immunity. However, upon mucosal delivery there is an increased chance of vaccine components being degraded or washed away prior to the induction of immunity. Thus, we added polyphosphazenes (PP) to our mucosal vaccine formulations. PP are synthetic polymers that form non-covalent complexes with other vaccine components, increasing their stability. Intranasally delivered FI-BRSV co-formulated with CpG ODN and PP performed better than FI-BRSV alone, or FI-BRSV formulated with either adjuvant individually, in terms of inducing protective immunity against BRSV in mice. Furthermore, mice that received intranasally-delivered FI-BRSV or BRSV F protein co-formulated with CpG ODN and PP developed higher levels of immunity and protection than mice that received parenterally delivered vaccines. Because of the similarities between BRSV and HRSV, co-formulation of intranasally delivered HRSV vaccines with CpG ODN and PP could prove important in the development of a safe vaccine against HRSV in humans.

Darbo tikslas: Atlikti PG-3 ir RS virusų infekcijų epidemiologinius tyrimus Lietuvos galvijininkystės ūkiuose, nustatyti pasyvaus ir aktyvaus imuniteto trukmę bei išaiškinti imuninio atsako galimybes, naudojant skirtingus vakcinacijos būdus. Darbo uždaviniai: 1. Nustatyti galvijų PG-3 ir RS virusų paplitimą Lietuvos galvijų ūkiuose. 2. Palyginti galvijų PG-3 ir RS virusinių infekcijų paplitimą su kitomis Lietuvos galvijų ūkiams aktualiomis virusinėmis ligomis. 3. Nustatyti galvijų paragripo-3 ir respiracinio sincitinio virusų paplitimo geografinius ypatumus. 4. Atlikti PG-3 ir RS virusinių infekcijų laboratorinės diagnostikos metodų palyginamąjį tyrimą. 5. Ištirti krekeninio imuniteto galvijų PG-3 ir RS virusams dinamiką ir trukmę bei jo įtaka veršelių sergamumui. 6. Atlikti vakcinacijos palyginamąjį tyrimą naudojant skirtingus įraumeninį (IM) ir įodinį (ID) vakcinacijos būdus ir bei skirtingas vakcinos dozes. Darbo mokslinė reikšmė ir naujumas: 1. Nustatytas PG-3 ir RS virusų paplitimas ir epidemiologiniai ypatumai Lietuvos galvijų ūkiuose. 2. Atliktas galvijų PG-3, RS, GIR ir GVD virusinių infekcijų paplitimo palyginamasis tyrimas. 3. Atliktas įvairių diagnostinių tyrimų palyginamasis įvertinimas. 4. Nustatytas galvijų PG-3 ir RS virusų serologinis paplitimas Lietuvos apskrityse. 5. Nustatyta pasyvaus krekeninio imuniteto trukmė galvijų PG-3 ir RS virusams bei jo įtaka veršelių sergamumui. 6. Nustatyta, kad ID vakcinacija nuo PG-3 ir RS virusinių infekcijų yra efektyvi... [toliau žr. visą tekstą] / The main objective of the present dissertation: To carry out epidemiological investigations of PI-3 and RS viral infections in the Lithuanian cattle farms, to establish the duration of passive and active immunity and to find out the possibilities of immune response using different routes of vaccination. Goals of the study: 1. To determine the prevalence of bovine PI-3 and RS viruses in the Lithuanian cattle farms. 2. To compare the prevalence of bovine PI-3 and RS viral infections with the prevalence of other cattle–relevant viral diseases at Lithuanian cattle farms. 3. To determine the geographical distribution patterns for PI-3 and RS viruses. 4. To conduct comparative analysis of laboratory diagnostic methods for PI-3 and RS viral infections. 5. To investigate the dynamics and duration of colostral immunity to bovine PI-3 and RS viruses and its influence on the morbidity of calves. 6. To conduct a comparative investigation of different routes of vaccination – intramuscular (IM) and intradermal (ID) – and different doses. Scientific importance and novelty of research: 1. The prevalence and epidemiological peculiarities of PI-3 and RS viruses in the Lithuanian cattle farms were determined. 2. A comparative analysis prevalence of bovine PI-3, RS, IBR and BVD viral infections was conducted. 3. A comparative assessment of various diagnostic investigations was performed. 4. The seroprevalence of bovine PI-3 and RS viral infections in the Lithuanian counties was determined. 5... [to full text]

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 &ldquo;desidratação leve&rdquo; (p&#61;0,033/ OR&#61; 11,25, IC: 1,809-41,834. Observou-se associações entre Mollicutes e a variável &ldquo;secreção nasal serosa&rdquo; (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 &ldquo;mild dehydration&rdquo; (p=0.033/OR=11.25; IC: 1.809-41.834). Associations between Mollicutes and &ldquo;serous nasal discharge&rdquo; (p=0.03) was also detected. In conclusion, S. intermedius was associated to &ldquo;nasal air flow&rdquo; (p=0.097). In conclusion bovine bronchopneumonia is multi-factorial disease. It is necessary, though, physical examination, microbiology and management and environment feature results

Orientador: Clarice Weis Arns / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T10:06:05Z (GMT). No. of bitstreams: 1 Spilki_FernandoRosado_D.pdf: 6085947 bytes, checksum: 7f34cc6e57c39e7467bfe62caa231676 (MD5) Previous issue date: 2007 / Resumo: O Vírus Respiratório Sincicial Bovino (BRSV) é uma causa importante de doença respiratória em bovinos. O BRSV é um membro da família Paramyxoviridae, subfamília Pneumovirinae, pertencendo ao gênero Pneumovirus. Nos últimos quinze anos, evidências sorológicas e de isolamento do vírus revelaram que o BRSV está circulando no Brasil, causando doença clinicamente evidente ou formas subclínicas da infecção. Na primeira parte do presente trabalho, seis diferentes linhagens celulares foram examinadas quanto a sua susceptibilidade à infecção pelo BRSV, levando em conta a variabilidade entre diferentes isolados e características de crescimento do vírus. Chicken embryo related cells (CER), e células CRIB (MDBK-resistentes à infecção pelo Vírus da Diarréia Viral Bovina, BVDV) foram as mais apropriadas à multiplicação do vírus. Ambas as linhagens permitiram o cultivo do vírus em títulos de até 105,5 DICC50 (Doses Infectantes para 50% dos Cultivos Celulares por 100 IlL)...Observação: O resumo, na íntegra poderá ser visualizado no texto completo da tese digital / Abstract: Bovine respiratory syncytial virus (BRSV) is a major cause of respiratory disease in young cattle. The virus is a member of the Paramyxoviridae family, Pneumovirinae subfamily, belonging to the Pneumovirus genus. During the last fifteen years, serological evidence and isolation of the virus revealed that BRSV is circulating in Brazil, causing clinically evident respiratory disease or subclinical fonns of the infection. In the first part of the present work, susceptibility of six different cell lines to BRSV'"li infection in regard to viral isolate variability and growth characteristics of the virus were examined. Chicken embryo related cells (CER), and bovine CRIB cells (a bovine viral diarrhea virus-resistant clone ofMDBK cells) showed to be the most appropriate for virus multiplication. Both cells provided infectious virus titres ofup to 105,5 TCID50 (50% tissue culture infective doses per 100 flL)...Note: The complete abstract is available with the full electronic digital thesis or dissertations / Doutorado / Microbiologia / Doutor em Genetica e Biologia Molecular

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 &ldquo;desidratação leve&rdquo; (p&#61;0,033/ OR&#61; 11,25, IC: 1,809-41,834. Observou-se associações entre Mollicutes e a variável &ldquo;secreção nasal serosa&rdquo; (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 &ldquo;mild dehydration&rdquo; (p=0.033/OR=11.25; IC: 1.809-41.834). Associations between Mollicutes and &ldquo;serous nasal discharge&rdquo; (p=0.03) was also detected. In conclusion, S. intermedius was associated to &ldquo;nasal air flow&rdquo; (p=0.097). In conclusion bovine bronchopneumonia is multi-factorial disease. It is necessary, though, physical examination, microbiology and management and environment feature results