Doctor of Philosophy / Department of Chemical Engineering / John R. Schlup / Jishu N. Shi / Vaccination is one of the most essential steps in controlling and preventing economically important infectious diseases in livestock. Vaccines need to be effective at producing a high level of immune responses that protect the animal from future encounters with infectious agents. Additional requirements for veterinary vaccines include safety, inexpensive components, and feasibility for large-scale production. These factors make emulsions attractive vaccine adjuvants. The use of emulsions as adjuvants (substances that help to amplify the immune responses to the antigen) has been explored for decades. However, emulsions are commonly produced with expensive and energy-demanding devices which impact the price of the adjuvant, therefore, affecting the price of the vaccines. This study examined low-energy emulsification methods to meet the requirements for a simple and low-cost vaccine manufacture that avoided utilizing complicated equipment. Spontaneous emulsification (SE) and phase inversion composition (PIC) was explored to formulate stable emulsions with nanometer droplet sizes. The study on the impact of oil composition on the formation of emulsions produced by SE revealed that addition of medium-chain triglycerides into the oil phase is beneficial for droplet size reduction and stability of emulsions. Box-Behnken design (BBD) was used to develop mathematical relationships between formulation variables and droplet size, polydispersity, zeta potential, and stability of emulsions formulated via SE. The BBD allowed the study of a simultaneous effect of multiple variables and formulate emulsions with certain physical characteristics, an effect that suggested that there was a more effective approach in designing complex systems like emulsions. New adjuvants containing mixtures of oils and surfactants were developed to produce emulsions with nanoscale droplet diameters and multiple water-in-oil-in-water structures via the PIC approach. The strong antibody responses and the absence of injection site side effects were observed in animals that received emulsion vaccines with experimental adjuvants. Additionally, inexpensive food-grade saponin extract was examined for stabilizing and increasing immunostimulatory activity of oil-in-water emulsion-based adjuvants. The adjuvants demonstrated high immune responses in pigs after co-administration with a subunit protein antigen.

Thesis (Ph. D.)--University of Melbourne, Dept. of Microbiology, 1998. / Includes bibliographical references.

Progressive Atrophic Rhinitis (PAR) is a serious complex disease of young swine characterized by sneezing, atrophy of the nasal turbinates, shortening and twisting of the snout and reduction in weight gain. Although the aetiology of the disease is complex, infection with the bacterium toxigenic Pateurella multocida, is considered essential. A dermonecrotic toxin (DNT) produced by toxigenic strains of type D P. multocida is central to the resorption of the nasal bone structures characteristic of the infection. The P. multocida DNT gene toxA has been previously cloned, sequenced and genetically manipulated in order to develop a vaccine for PAR. These earlier studies demonstrated that DNT-specific antibodies produced in pigs by vaccination with the purified genetically inactivated DNT derivative (toxoid) resulted in the protection of the animals against experimentally induced PAR. An alternative approach to using a subunit vaccine for PAR is to express the toxoided gene from P. multocida either from the chromosome or a plasmid thus providing a live vaccine that could present to the porcine immune system a full spectrum of bacterial antigens in addition to the DNT. (For complete abstract open document)

Bovine Neonatal Pancytopenia (BNP) is a disease of calves, characterised by peripheral blood and bone marrow depletion, which emerged in Europe in 2007. A strong epidemiological association between BNP and the administration of a particular inactivated Bovine Viral Diarrhoea (BVD) vaccine (Pregsure BVD, Pfizer Animal Health) to the dams of affected calves has been reported. Early studies suggested that BNP is mediated by the transfer of alloantibodies in colostrum and that these alloantibodies recognise major histocompatibility complex (MHC) class I molecules. This led to the hypothesis that Pregsure contains bovine MHC I molecules, originating from the MDBK line cell used in vaccine production, and that this is responsible for the generation of alloantibodies in particular cows injected with the vaccine. This project aimed to investigate the mechanisms by which BNP arises and develops. In particular to gain an understanding of the molecular basis of the syndrome and how this influences the number of cows and calves affected and the specificity of the pathology for the haematopoietic system. Haematological analysis of clinically normal calves born on a BNP-affected farm demonstrated that 15% of calves had profoundly abnormal haematology and could be described as affected by subclinical BNP. BNP was reproduced experimentally by feeding pooled colostrum to neonatal calves, confirming the role of colostrum in mediating the condition. Detailed analysis of serial haematology and bone marrow pathology from these calves demonstrated variable alloantibody damages to different haematopoietic lineages. In vitro cellular assays using a panel of MHC I-defined bovine leukocyte cell lines and mouse cells individually transfected with the MDBK-MHC I alleles demonstrated that Pregsure vaccinated cows have significantly higher titres of functionally active MHC I alloantibodies than BVDV unvaccinated cows or cows vaccinated with alternative BVDV vaccines. The alloantibody response was found to be heterogeneous in individual Pregsure vaccinated cows. MHC I expression levels on peripheral blood and bone marrow cells, assessed by flow cytometry, was shown to correlate with levels of in vitro and in vivo alloantibody damage. Overall, the results of this project demonstrate that the pathogenesis of BNP is mediated by the transfer of MHC I-specific alloantibodies via colostrum that cause rapid destruction of peripheral blood and bone marrow cells, and which is dependent on the titre of alloantibody produced by an individual cow, its specificity for the MHC I alleles present, and density of MHC I expression on the specific cells.

Historically, the causative agent of infectious coryza has been identified as the NAD requiring bacterium Haemophilus paragallinarum and the implementation of an intensive vaccination program led to the effective control of this contagious upper respiratory infection. More recently, however, a decline in the protective capacity of a vaccine conditioned immune response was noted, with a number of contributing factors, including the emergence of a fast-growing NAD-independent bacterium, which has largely replaced the traditional NAD-dependent variety. As such, accurate, reproducible methods for determining and continually monitoring the type of infecting bacteria was necessitated. To address this need, strains of H. paragallinarum were evaluated according to both their phenotypic and their genotypic properties, in a combination serodiagnostic approach. A data bank of NAD-dependent H. paragallinarum reference strain and field isolate serovar-specific fingerprints was established on both a whole cell and outer membrane protein level. Visual comparative analysis of the qualitatively and quantitatively similar outer membrane protein patterns of all strains of NAD independency studied with the formulated data bank, indicate that the NAD-independent strains displayed profiles typical of serovar C-3. The outer membrane proteins have been identified as putative virulence determinants and, as such, were characterised according to their surface location, susceptibility to heat modification, functional role as endotoxins, sequence homology to structural membrane counterparts, and finally, their ability to induce an immune response. These studies represent novel efforts and form the foundation for identifying those antigens responsible for maintaining an infection in the host milieu. Ribotype analysis served as an adjunct to phenotypic observations, with the local NAD-independent field isolates being identified as serotype A. These contradictory outcomes call for the creation of a set of reference strains specific for NAD-independent isolates. The identification of restriction fragment length polymorphisms in the conserved 16S rRNA gene sequences indicate the potential application of this method for type assignment, requiring the recognition of a battery of versatile restriction enzymes to generate serovar-specific polymorphic profiles. The complexity of serotype allocation demands that a combination approach in which genotypic analyses complement phenotypic-based methods of haemagglutination inhibition and outer membrane protein profiling. The groundwork for implementation of such a system has been accomplished. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1998.

Orientador: Angelo Berchieri Junior / Coorientador: Hélio José Montassier / Banca: Rosângela Zacarias Machado / Banca: Luiz Felipe Caron / Banca: Raphael Lucio Andreatti Filho / Banca: Marcelo Brocchi / Resumo: Salmonella Enteritidis (SE) causa doença transmitida por alimentos (DTA) em humanos. Carne de frango e ovos frequentemente estão associados a esses casos. O controle da infecção em aves, baseia-se em medidas de biossegurança, incluindo-se a vacinação. Tem sido comum a utilização de vacinas vivas (VV) e inativadas (Bacterinas - BA), porém pouco se sabe sobre os mecanismos imunes desencadeados pelas vacinas contra SE. Neste estudo, utilizaram-se quatroprogramas vacinais (VV; VV+VV; BA; VV+BA) em galinhas leves de variedade branca para postura de ovos de mesa, vacinadas com 5 e/ou 25 dias de vida, e desafiadas aos 45 dias de vida com SE. No dia anterior à infecção (1 DAI) e 1, 6 e 9 dias pós-infecção (DPI), cinco aves/grupo foram sacrificadas para amostragem. A população de linfócitos CD4+ e CD8+ foi avaliada por imuno-histoquímica em tonsilas cecais e fígado; citocinas foram quantificadas por RT-qPCR em tempo real em tonsilas cecais e baço; níveis de IgG e IgM foram mensurados por ELISA no soro e IgA em lavado intestinal. Os níveis de imunoglobulina (IgG, IgM e IgA) estavam significativamente mais altos em aves vacinadas com BA, do 1 DAI ao 9 DPI, em comparação aos grupos vacinados somente com VV. Os níveis de IFN-γ, na tonsila cecal, eram similares em todos os grupos após o desafio. Antes do desafio (1 DAI), IL-10 foi altamente expressa em baço de aves que receberam somente BA (25 dias de vida), sugerindo o desenvolvimento de resposta por linfócitos T CD4+ auxiliar 2 (Ta2), reforçado pelos altos níveis de IgG encontrados neste grupo (p<0.05). Os níveis de TGF-β4 e das citocinas pró-inflamatórias IL-6 e TNFSF15 eram bastante elevados no grupo que recebeu VV+VV. A vacinação por via oral com VV aumentou significativamente o fluxo de linfócitos T CD8+ para as tonsilas cecais após o desafio. Isso poderia... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Salmonella Enteritidis (SE) causes foodborne infection in humans. Poultry meat and eggs are frequently associated with these cases. The control of this bacterium is based on sanitary measures and mainly, vaccination of chicken flocks. Vaccine programs are used worldwide to control SE in poultry flocks. Live (LV) and killed vaccines (KV) are often combined, although few studies regarding immune mechanisms developed by SE vaccines are available. In this work, four vaccine programs were studied (LV; LV+LV; KV; LV+KV) in white layer-hens vaccinated at 5 and/or 25 days-old and challenged at 45 days-old with SE. At 1 day before (dbi) and 1, 6 and 9 days post-infection (DPI), five birds/group were sacrificed for sampling. The population of CD4+ and CD8+ T cells was evaluated by immunohistochemistry in caecal tonsils and liver, cytokines were quantified by real time RT-qPCR in caecal tonsils and spleen; IgG, IgM and IgA levels were measured by ELISA in serum and the latter in intestinal washes. The immunoglobulin levels (IgG, IgM and IgA) were significantly higher in birds vaccinated with KV, from 1 dbi to 9 DPI, than in groups that received only LV. In caecal tonsils, IFN-γ levels were similar in all groups after challenge. Before challenge (1 dbi), IL-10 was highly expressed in spleen of birds that received only KV (25 days-old), suggesting the development of the T CD4+ helper 2 (Th2) type of immune response, reinforced by high IgG levels against SE seen in this group (p<0.05). TGF-β4 and the proinflammatory cytokines IL-6 and TNFSF15 were higher in caecal tonsils in the group that received LV+LV. Vaccination by oral route with LV clearly increased the influx of CD8+ T cells in caecal tonsils after challenge. This could be correlated with the better control of SE noticed in groups that received at least one dose of LV, including... (Complete abstract click electronic access below) / Doutor

Orientador: Hélio José Montassier / Banca: Ricardo Luiz Moro de Sousa / Banca: Samir Issa Samara / Resumo: A bronquite infecciosa das galinhas (BIG) é uma doença infecciosa causada pelo coronavírus aviário (vírus da bronquite infecciosa - VBI) que está amplamente disseminada entre as criações avícolas comerciais na maior parte do mundo. Há atualmente no Brasil, uma predominância de infecções causadas por estirpes do VBI classificadas no genótipo variante (BR-I), que revelam diferenças marcantes de antigenicidade com relação à estirpe vacinal Massachusetts, rotineiramente usada em nosso país. Isso resulta em uma baixa imunidade-cruzada e consequentemente em um menor nível de proteção contra isolados de campo do genótipo BR-I. Dessa forma, os objetivos principais do presente estudo foram formular uma vacina experimental inativada com, uma estirpe IBVPR-05 variante do genótipo BR-1 do VBI acrescida de um novo adjuvante oleoso; testá-la após a administração, com 1 dia de idade, da vacina comercial atenuada com estirpe Massachusetts, avaliando-se as respostas imunes humorais e cito-mediadas, bem como o estado de proteção ao desafio com essa estirpe variante do VBI através da avaliação histopatológica e quantificação absoluta da carga viral presente na traqueia e rins de aves vacinadas e desafiadas com a variante. Os resultados deste estudo demonstraram que esse esquema imunoprofilático (vacinação das aves com 1 dia de idade com vacina atenuada comercial e revacinação com 14 dias de idade com vacina inativada experimental homóloga) induziu aumentos significativos nos níveis de anticorpos lacrimais e séricos do isótipo IgG anti-VBI e também na expressão dos genes relacionados às respostas imunes cito-mediadas, sobretudo o da cadeia  do CD8 e da Granzima A, nas aves vacinadas, que se mostraram associados com a diminuição de lesões histológicas e uma menor carga viral na traqueia e rins nas aves vacinadas e desafiadas. Concluiu-se que as respostas imunes humoral e celular de memória conferidas ... / Abstract: The infectious bronchitis (BIG) is an infectious disease caused by avian coronavirus (infectious bronchitis virus - IBV) that is widespread among commercial poultry flocks in the world. There are currently in Brazil, a predominance of infections caused by strains of IBV classified in variant genotype (BR-I), which reveal striking differences in antigenicity with regard to the vaccine strain Massachusetts, routinely used in this country. The consequence is a low cross-immunity and lower level of protection against Brazilian field isolates of genotype BR-I. Thus, the main objectives of this study were to formulate an experimental inactivated vaccine with a variant strain of IBV previously characterized as genotype BR-I and added of a new oil adjuvant and test it after an administration, at 1 day old, of a commercial attenuated Massachusetts vaccine, followed by the evaluation of humoral and cellular immune (CMI) responses, as well as the state of protection upon challenge with this variant strain of IBV by histopathological examination and absolute quantification of viral load present in the trachea, and kidneys of these birds vaccinated and challenged with this variant strain. The results of this study demonstrated that this immunoprophylactic approach (vaccination of birds with 1 day old with attenuated commercial vaccine and revaccination at 14 days of age with annual experimental inactivated vaccine) was able to elicit significant increases in the serum and tear levels of anti-IBV antibodies of IgG isotype, and also in the expression of CMI genes, especially of CD8  chain and Granzyme A in the vaccinated birds, which were associated with decreased histological lesions and reduced viral load in the trachea and kidney of vaccinated and challenged birds. It was concluded that humoral and cellular memory immune responses conferred by vaccination with this Brazilian variant strain of IBV combined to a previous Massachusets ... / Mestre

Mycoplasma hyopneumoniae is the causative agent of the respiratory disease Swine Enzootic Pneumonia, a mild chronic lower respiratory tract infection that affects pig populations world wide. The disease causes decreased growth rates and poor feed conversion in infected pigs and results in significant economic losses. While several swine enzootic pneumonia vaccines arc available, none are totally effective. These current vaccines are based on bacterins or cell fractions. As yet, no commercial vaccines composed of recombinant subunits are available. Prior to the commencement of this study, three candidate vaccinc antigens had been identified in this laboratory, from the Australian M. hyopneumoniae field isolate Beaufort. The three proteins (48 kDa, 52 kDa and 74 kDa) reacted with antibody secreting cell probes, derived from hyper-immune swine lung tissue, indicating they are important in the local antibody response to M. hyopneumoniae infection. (For complete abstract open document)

Thesis (M.S.)--University of Missouri-Columbia, 2006. / "August 2006" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.

Made available in DSpace on 2014-06-11T19:33:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-02-22Bitstream added on 2014-06-13T20:44:56Z : No. of bitstreams: 1 penhafilho_rac_dr_jabo.pdf: 670109 bytes, checksum: 048e4ba89a0df3d41f36db27a93c0f9b (MD5) / Salmonella Enteritidis (SE) causa doença transmitida por alimentos (DTA) em humanos. Carne de frango e ovos frequentemente estão associados a esses casos. O controle da infecção em aves, baseia-se em medidas de biossegurança, incluindo-se a vacinação. Tem sido comum a utilização de vacinas vivas (VV) e inativadas (Bacterinas - BA), porém pouco se sabe sobre os mecanismos imunes desencadeados pelas vacinas contra SE. Neste estudo, utilizaram-se quatroprogramas vacinais (VV; VV+VV; BA; VV+BA) em galinhas leves de variedade branca para postura de ovos de mesa, vacinadas com 5 e/ou 25 dias de vida, e desafiadas aos 45 dias de vida com SE. No dia anterior à infecção (1 DAI) e 1, 6 e 9 dias pós-infecção (DPI), cinco aves/grupo foram sacrificadas para amostragem. A população de linfócitos CD4+ e CD8+ foi avaliada por imuno-histoquímica em tonsilas cecais e fígado; citocinas foram quantificadas por RT-qPCR em tempo real em tonsilas cecais e baço; níveis de IgG e IgM foram mensurados por ELISA no soro e IgA em lavado intestinal. Os níveis de imunoglobulina (IgG, IgM e IgA) estavam significativamente mais altos em aves vacinadas com BA, do 1 DAI ao 9 DPI, em comparação aos grupos vacinados somente com VV. Os níveis de IFN-γ, na tonsila cecal, eram similares em todos os grupos após o desafio. Antes do desafio (1 DAI), IL-10 foi altamente expressa em baço de aves que receberam somente BA (25 dias de vida), sugerindo o desenvolvimento de resposta por linfócitos T CD4+ auxiliar 2 (Ta2), reforçado pelos altos níveis de IgG encontrados neste grupo (p<0.05). Os níveis de TGF-β4 e das citocinas pró-inflamatórias IL-6 e TNFSF15 eram bastante elevados no grupo que recebeu VV+VV. A vacinação por via oral com VV aumentou significativamente o fluxo de linfócitos T CD8+ para as tonsilas cecais após o desafio. Isso poderia... / Salmonella Enteritidis (SE) causes foodborne infection in humans. Poultry meat and eggs are frequently associated with these cases. The control of this bacterium is based on sanitary measures and mainly, vaccination of chicken flocks. Vaccine programs are used worldwide to control SE in poultry flocks. Live (LV) and killed vaccines (KV) are often combined, although few studies regarding immune mechanisms developed by SE vaccines are available. In this work, four vaccine programs were studied (LV; LV+LV; KV; LV+KV) in white layer-hens vaccinated at 5 and/or 25 days-old and challenged at 45 days-old with SE. At 1 day before (dbi) and 1, 6 and 9 days post-infection (DPI), five birds/group were sacrificed for sampling. The population of CD4+ and CD8+ T cells was evaluated by immunohistochemistry in caecal tonsils and liver, cytokines were quantified by real time RT-qPCR in caecal tonsils and spleen; IgG, IgM and IgA levels were measured by ELISA in serum and the latter in intestinal washes. The immunoglobulin levels (IgG, IgM and IgA) were significantly higher in birds vaccinated with KV, from 1 dbi to 9 DPI, than in groups that received only LV. In caecal tonsils, IFN-γ levels were similar in all groups after challenge. Before challenge (1 dbi), IL-10 was highly expressed in spleen of birds that received only KV (25 days-old), suggesting the development of the T CD4+ helper 2 (Th2) type of immune response, reinforced by high IgG levels against SE seen in this group (p<0.05). TGF-β4 and the proinflammatory cytokines IL-6 and TNFSF15 were higher in caecal tonsils in the group that received LV+LV. Vaccination by oral route with LV clearly increased the influx of CD8+ T cells in caecal tonsils after challenge. This could be correlated with the better control of SE noticed in groups that received at least one dose of LV, including... (Complete abstract click electronic access below)