Thesis (M.Sc.)--University of Adelaide, 1952. / Typewritten copy.

Pasteurella multocida é um importante patógeno para suínos, causando rinite atrófica progressiva, pneumonia, pleurite, e septicemia. Para implantação de estratégias de controle e prevenção desta infecção, torna-se necessário o conhecimento a respeito do perfil de disseminação do agente em condições naturais e em diferentes tipos de sistema de produção. Tendo em vista a inexistência de técnicas sorológicas padronizadas para esta finalidade na espécie suína, o presente estudo teve por objetivos avaliar em um grupo de 90 animais a influência de diferentes sítios de coleta de amostra na detecção de animais portadores de P. multocida através da PCR e comparar estes dados a detecção de anticorpos na espécie suína através de um kit de ELISA comercial registrado para detecção de anticorpos contra P. multocida em aves. Foram coletados suabes de cavidade nasal, suabes de tonsila e sangue de 90 animais em idade de abate provenientes de dois sistemas de produção de suínos. Dentre os 90 animais, 14 (15,55%) foram positivos para detecção de P. multocida em suabes nasais e nenhum foi positivo em suabe de tonsila. Através do ELISA, 25 (27,77%) animais apresentaram anticorpos contra o agente. Através da análise de comparação de proporção, houve diferença significativa em relação à metodologia de diagnóstico nos diferentes testes realizados considerando-se valor de p < 0,0001 e intervalo de confiança de 95%. Ou seja, a freqüência de positivos foi significativamente maior no teste de ELISA, seguido pela reação em cadeia pela polimerase em suabes nasais, sugerindo que a cavidade nasal é o sitio primário de colonização dos animais por este agente e que o ELISA testado pode ser facilmente adaptado para avaliação do perfil sorológico do agente na espécie suína. / Pasteurella multocida is an important pathogen for pigs, causing progressive atrophic rhinitis, pneumonia, pleurisy, and septicemia. For implementation of strategies to control and prevent infections, it is necessary to know about the profile of agent spread in natural conditions and in different types of production system. Given the lack of standardized serological techniques for this purpose, this study aims to evaluate the influence of different methods and sites of sample collection in the detection of animals with P. multocida by polymerase chain reaction (PCR) and ELISA. We evaluated different pairs of primers specific for the agent and the reaction that have lower detection threshold will be used in the evaluation of the study sites. Swabs were collected from the nasal cavity, tonsil and also blood of 90 animals. Among these, 14 (15.55%) were positive for Pasteurella multocida by polymerase chain reaction (PCR), all of which were nasal swabs. There are no positive animals among the tonsil swabs. In the ELISA, 25 (27.77%) were positive, and of these, three (3.33%) were positive in both the polymerase chain reaction and the ELISA and the remaining 22 (24.44%) was positive by ELISA and negative by polymerase chain reaction. Using comparison of proportion analysis, was observed a significant differences in the methodology of diagnosis in different tests considering p-value <0.0001 and a confidence interval of 95%. Concluding, the frequency of positives was significantly higher in ELISA than by the polymerase chain reaction.

Pasteurella multocida é um importante patógeno para suínos, causando rinite atrófica progressiva, pneumonia, pleurite, e septicemia. Para implantação de estratégias de controle e prevenção desta infecção, torna-se necessário o conhecimento a respeito do perfil de disseminação do agente em condições naturais e em diferentes tipos de sistema de produção. Tendo em vista a inexistência de técnicas sorológicas padronizadas para esta finalidade na espécie suína, o presente estudo teve por objetivos avaliar em um grupo de 90 animais a influência de diferentes sítios de coleta de amostra na detecção de animais portadores de P. multocida através da PCR e comparar estes dados a detecção de anticorpos na espécie suína através de um kit de ELISA comercial registrado para detecção de anticorpos contra P. multocida em aves. Foram coletados suabes de cavidade nasal, suabes de tonsila e sangue de 90 animais em idade de abate provenientes de dois sistemas de produção de suínos. Dentre os 90 animais, 14 (15,55%) foram positivos para detecção de P. multocida em suabes nasais e nenhum foi positivo em suabe de tonsila. Através do ELISA, 25 (27,77%) animais apresentaram anticorpos contra o agente. Através da análise de comparação de proporção, houve diferença significativa em relação à metodologia de diagnóstico nos diferentes testes realizados considerando-se valor de p < 0,0001 e intervalo de confiança de 95%. Ou seja, a freqüência de positivos foi significativamente maior no teste de ELISA, seguido pela reação em cadeia pela polimerase em suabes nasais, sugerindo que a cavidade nasal é o sitio primário de colonização dos animais por este agente e que o ELISA testado pode ser facilmente adaptado para avaliação do perfil sorológico do agente na espécie suína. / Pasteurella multocida is an important pathogen for pigs, causing progressive atrophic rhinitis, pneumonia, pleurisy, and septicemia. For implementation of strategies to control and prevent infections, it is necessary to know about the profile of agent spread in natural conditions and in different types of production system. Given the lack of standardized serological techniques for this purpose, this study aims to evaluate the influence of different methods and sites of sample collection in the detection of animals with P. multocida by polymerase chain reaction (PCR) and ELISA. We evaluated different pairs of primers specific for the agent and the reaction that have lower detection threshold will be used in the evaluation of the study sites. Swabs were collected from the nasal cavity, tonsil and also blood of 90 animals. Among these, 14 (15.55%) were positive for Pasteurella multocida by polymerase chain reaction (PCR), all of which were nasal swabs. There are no positive animals among the tonsil swabs. In the ELISA, 25 (27.77%) were positive, and of these, three (3.33%) were positive in both the polymerase chain reaction and the ELISA and the remaining 22 (24.44%) was positive by ELISA and negative by polymerase chain reaction. Using comparison of proportion analysis, was observed a significant differences in the methodology of diagnosis in different tests considering p-value <0.0001 and a confidence interval of 95%. Concluding, the frequency of positives was significantly higher in ELISA than by the polymerase chain reaction.

Typescript (photocopy). / Digitized by Kansas Correctional Industries

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

A key bacterial virulence factor is the ability to acquire the micronutrient iron, required by a vast majority of micro-organisms for survival and proliferation within their hosts. The work described here focuses on acquisition of iron in Pasteurella multocida serotype A:3, a Gram-negative opportunistic pathogen that causes pneumonic pasteurellosis in cattle, a severe respiratory infection of significant economic burden and welfare concern. P. multocida A:3 acquires iron primarily from the host iron-chelating molecule transferrin through the expression of specific outer membrane receptors. The correlation between up-regulation of these receptors and of other iron-regulated outer membrane proteins (IROMPs) and an increase in bacterial virulence has been noted elsewhere. To date, there has been no systems analysis of the metabolic processes of iron acquisition published for any bacterial species. The work described here used a systems approach involving elementary flux modes analysis to derive a computational model of iron acquisition and reveal a number of key findings on the mechanisms of iron acquisition and links between iron uptake, glucose metabolism and protein synthesis. This in silico model was subjected to experimental validation through a range of in vitro experiments designed to investigate the links between iron restriction and growth and metabolism of P. multocida. This novel approach was only possible after the development and optimisation of a number of assays to measure key model parameters.

El hierro y el zinc son micronutrientes esenciales para el crecimiento normal de la gran mayoría de microorganismos, siendo los mecanismos de captación de estos cationes divalentes elementos fundamentales para que las bacterias patógenas sean capaces de promover un proceso infectivo. Además, las proteínas receptoras de hierro se localizan en la membrana externa de las bacterias Gram negativas, lo cual las hace candidatas a ser empleadas para el diseño de vacunas.En este marco, el objetivo de la presente tesis doctoral ha sido profundizar en el estudio de los mecanismos de captación de cationes divalentes de Pasteurella multocida, una bacteria patógena que afecta a una gran variedad de animales originando importantes pérdidas económicas en el sector ganadero.En este estudio se ha identificado un receptor de hierro de 60 kDa, al que se ha denominado HbpA, que presenta un mecanismo de regulación distinto al utilizado comúnmente por las bacterias, ya que es independiente del sistema Fur, determinándose que su regulación depende de Fe2+, Mn2+ y hemina. Se ha demostrado que el gen que codifica esta proteína presenta un corrimiento programado de lectura que da lugar a un derivado truncado de 40 kDa. Se ha caracterizado así mismo la función de esta proteína, determinándose que une tanto hemina como hemoglobina, y que la capacidad de unión a ambas se mantiene en el derivado truncado de la proteína. Además, se ha estudiado la antigenicidad de la proteína HbpA, así como su efecto inmunogénico, demostrándose que la proteína HbpA y su derivada truncada son antigénicas, pero no se obtiene protección cuando se administra dicha proteína entera en experimentos de enfrentamiento utilizándose un modelo experimental animal de ratón.Por lo que se refiere a los mecanismos de incorporación de zinc de P. multocida, desconocidos hasta el presente, se han caracterizado los genes de captación de zinc de alta afinidad (znuABC), determinándose que existe una separación de 820 kb entre los genes znuA y znuCB y que los genes znuC y znuB forman parte de una misma unidad transcripcional. A diferencia de lo que ocurre en Escherichia coli y en otras bacterias, en las cuales la proteína Zur regula la expresión de este sistema de captación de zinc, en P. multocida no se ha encontrado una proteína homóloga a dicha proteína Zur. No obstante, se ha podido establecer que en este patógeno es la proteína Fur la responsable de la regulación de las unidades transcripcionales znuA y znuCB, juntamente con los iones Zn2+ y Fe2+. Finalmente, mediante la construcción de mutantes znuA y znuC, también se ha demostrado que estos genes son imprescindibles para la virulencia de P. multocida. / Iron and zinc are essential for the normal growth of almost all the microorganisms, being the uptake mechanisms of these cations elemental for the infective process of pathogenic bacterial. Moreover, in Gram negative bacterial, the iron receptor proteins are located in the outer membrane, making them good candidates in the vaccine design.In this framework, the doctoral thesis aim have been to go deeply into the study of divalent cations uptake mechanisms in Pasteurella multocida, a pathogenic bacterial that has an effect on a wide variety of animals, originating important economic losses in the stock sector.In this study it has been identified a 60-kDa iron receptor, named HbpA, which regulation mechanism is different to that used commonly by the bacterial, because it is Fur pathway independent, being its regulation Fe2+, Mn2+ and haemin dependent. It has being demonstrated that the codifying gene of this protein has a programmed translational frameshift giving rise to a 40 kDa protein. The function of this protein has been characterized as well, demonstrating that it binds both haemin and haemoglobin and this union capacity is maintained in the truncated derivate protein. Furthermore, the antigenicity and its potential protective ability have been studied, showing that HbpA and its truncated derivate are antigenic, but there is non-protective effect when this protein is given in challenge experiments using a mouse animal model. In reference to the zinc uptake pathways in P. multocida, unknown until the present, it has been characterized the high affinity uptake system (znuABC) genes, showing that there is a distance of 820 kb between znuA and znuCB genes, and znuC and znuB are part of the same transcriptional unit. Contrary to what happens in Escherichia coli and other bacterial, in which the Zur protein controls the expression of this high affinity uptake system; in P. multocida has not been found a homolog to this Zur protein. Nevertheless, it could be established that, in this pathogen, the responsible protein of the znuA and znuCB control is the Fur protein, joined with the Zn2+ and Fe2+ ions. Finally, using znuA and znuC mutants, it has also been demonstrated that these genes are essential for the virulence in P. multocida.

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)

Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1993. / Vita. Abstract. Includes bibliographical references (leaves 104-120). Also available via the Internet.

Thesis (Ph.D.) - University of Queensland, 2002. / Includes bibliography.