Tesis sobre el tema "Pasteurella multocida"

Realiza un análisis pangenómico y filogenómico para estudiar la diversidad genómica de esta bacteria y determinar si las cepas de distintos hospederos y enfermedades contienen diferencias en cuanto a contenido genético. Según el análisis, Pasteurella multocida posee un pangenoma abierto de 4881 genes y un genoma core de 1205 genes (25%). El genoma accesorio y único (75%) contienen mayormente secuencias profago y proteínas de función desconocida, junto con la alta presencia de recombinación en el genoma core (45%) son los principales generadores de diversidad mediante transferencia horizontal de genes y recombinación homóloga. El análisis filogenómico del genoma core y pangenoma muestra el agrupamiento de cepas asociadas a enfermedades específicas sugiriendo una especialización en esta bacteria con presencia de genes de manera diferencial en cepas asociadas a enfermedades como septicemia hemorrágica, cólera aviar, pasteurelosis neumónica y relacionados a infecciones en humanos. Las cepas de P. multocida asociadas a cólera aviar poseen operones relacionados a metabolismo de carbohidratos como L-fucosa, D-alosa, citrato, L-arabinosa, tagatosa y galactitol, en tanto que cepas asociadas a infecciones neumónicas presentan un operón de trehalosa, las cepas asociadas a septicemia hemorrágica poseen genes de biosíntesis de ipopolisacáridos, adherencia celular y defensa a macrófagos, y algunas cepas relacionadas a humanos carecen de conjuntos de genes asociados principalmente a transporte y metabolismo de carbohidratos. Estas diferencias en contenido y función genética pueden ser responsables de la variada patogénesis de P. multocida relacionado con la colonización e invasión en los primeros momentos de la infección.
Tesis

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.

A Pasteurella multocida é o agente causador da pasteurelose pulmonar dos suínos, sendo comum a ocorrência dessa infecção associada com o Mycoplasma hyopneumoniae no curso da pneumonia enzoótica. O agente é encontrado em pulmões de várias espécies animais e no homem. A infecção de suínos com o circovírus suíno tipo 2 (PCV2) causa uma forma de infecção sistêmica com profundos efeitos no sistema linfóide, causando prejuízos aos processos normais de defesa e facilitando a instalação de infecções secundárias, incluindo a pneumonia enzoótica complicada pela Pasteurella multocida. O objetivo deste trabalho foi isolar e caracterizar amostras de P. multocida obtidas de suínos com sinais clínicos compatíveis com os da circovirose e de animais de frigorífico quanto ao seu perfil bioquímico, capsular e sensibilidade a antimicrobianos. Foram utilizados 453 materiais (pulmões e suabes das lesões pulmonares), resultando em 115 isolados bacterianos de 79 pulmões. Paralelamente, procurou-se avaliar a eficiência do isolamento da bactéria a partir de dois sítios de amostragem no pulmão: do exsudato bronquial e da superfície de corte pulmonar. Numa amostragem dos casos, procurou-se estabelecer a relação entre a infecção com PCV2 e as lesões pulmonares induzidas pela P. multocida. Numa análise do grau de pneumonia foram classificadas 38 lesões como leves, 19 como médias e 15 graves. Na análise bacteriológica, todas as amostras foram positivas para o teste da catalase e oxidase e negativas para o citrato e H2S, e 11,3% foram negativas ao teste do indol. Utilizando-se os substratos sorbitol, manitol, trealose, maltose e arabinose foram observados seis perfis distintos. O tipo capsular A esteve presente em 93,91% das amostras e o tipo D em 6,09%. Nenhuma das amostras possuía o gene codificador da toxina dermonecrótica (toxA) e a resistência antimicrobiana foi baixa e a oxitetraciclina o princípio ativo com maior resistência 15,65%.
Pasteurella multocida is the etiologic agent of swine respiratory pasteurellosis and the infection occurs specially associated with Mycoplasma hyopneumoniae in the course of enzootic pneumonia. The agent can be isolated from the lung of several animal species and man. Infection of pigs with porcine circovirus (PCV2) causes a systemic infection with deep effects on the lymphoid system (porcine multisystemic wasting syndrome, PMWS), resulting in impairment of the normal defense processes and facilitating secondary infections, including enzootic pneumonia complicated with Pasteurella multocida. The objective of the present work was to isolate and characterize strains of P. multocida obtained from pigs with clinical signs compatible with PMWS and from slaughter pigs, considering biochemical profile, capsular typing and antibiotic sensitivity testing. A total of 453 materials (lungs and swabs from lung lesions) were used, resulting in 115 bacterial isolates from 79 lungs. At the same time, the efficiency of the isolation of the bacteria from two different sites in the lung was assessed: bronchial exudate and the cut surface of the lung. In a sample of the cases it was tried to establish the relationship between PCV2 infection and lung lesions induced by P. multocida. Analyzing degrees of pneumonia, 38 lesions were classified as mild, 19 as intermediate and 15 as severe. In the bacteriological analysis, all strains were positive for catalase and oxidase, and negative for citrate and H2S, and 11.3 were negative in the indole test. Using sugar fermentation (sorbitol, manitol, trealose, maltose and arabinose) six different profiles were established. Capsular type A was diagnosed in 93.91% of the samples and type D in 6.09%. All strains were negative for the gene codifying dermonecrotic toxin (toxA). Antimicrobial resistance was low, and tetracycline was the product showing higher resistance (15.65%).

A 4.0 Kb (2.64 Mdal) plasmid was isolated from a fowl cholera strain of Pasteurella multocida (the Larsen strain) by alkaline lysis and cesium chloride purification. The plasmid, designated pLAR-1, was characterized in terms of its size and restriction sites. The restriction patterns produced by fourteen endonucleases were used to generate a restriction map. Five restriction enzymes cleaved the plasmid at multiple sites. Two enzymes, Bgl II and Sal I had unique sites on pLAR-1. Twelve of the fifty six strains of P. multocida surveyed contained plasmids of different sizes which hybridized to pLAR-1. Strains containing homologous plasmids were variable in serotype, dermonecrotoxin production, and origin (both in terms of the host and locale). pLAR-1 did not encode any of the enzymes necessary for the biochemical pathways contained within the API-20E strip or siderophore production. pLAR-1 was cloned into the BamH I site of pBR322. Resultant clones were approximately 8.363 Kb in length, ampicillin resistant and tetracycline sensitive. The pLAR-1
Master of Science

Pasteurella multocida, apesar de ser uma bactéria que compõe a microbiota respiratória, sob algumas circunstâncias pode manifestar-se como um agente patogênico primário ou secundário, causando doença em aves e outros animais. Como agente primário, P. multocida leva a grandes perdas econômicas, causando cólera em aves, rinite atrófica em suínos e septicemia hemorrágica em bovinos e bubalinos. P. multocida é uma espécie heterogênea, e a patogenicidade dos isolados pode ser amplamente variável. A suscetibilidade do hospedeiro à essas cepas varia consideravelmente entre espécies. Inoculações experimentais de P. multocida em camundongos e aves são comumente usadas para avaliar a patogenicidade de diferentes cepas, mas os resultados são geralmente subjetivos e pouco mensuráveis. O objetivo deste trabalho foi estabelecer uma nova metodologia para classificar a patogenicidade de cepas de P. multocida, através da formulação de um índice padrão. Para determinar esse índice, foram usadas 97 amostras de P. multocida, isoladas de cólera em aves e rinite em suínos. Cem microlitros (105 UFC) de uma cultura bacteriana contendo 106 UFC/mL, de cada isolado de P. multocida foram inoculados, por via intraperitoneal, em 10 pintos de um dia. Além da mortalidade causada pela inoculação, o tempo de morte e as lesões macroscópicas foram avaliados. Diferenças significativas foram observadas entre isolados de aves e suínos em relação aos índices de patogenicidade. O número de lesões e a porcentagem de bactérias recuperadas dos animais inoculados também variaram de acordo com a origem do isolado. A partir dos indices observados, os isolados foram distribuídos em três classes de patogenicidade: alta, média e baixa. O índice de patogenicidade desenvolvido neste trabalho permite a mensuração e classificação da patogenicidade de isolados de P. multocida e pode ser uma alternativa aos modelos subjetivos de triagem de patogenicidade usados até o momento.
Pasteurella multocida, despite being a bacteria that makes up the respiratory microbiota, under some circumstances can manifest as primary or secondary pathogenic agent, causing disease in birds and other animals. As primary agent, P. multocida leads to large economic losses, causing fowl cholera in birds, atrophic rinitis in swine and haemorragic septicemia in cattle and buffalos. P. multocida is a heterogeneous specie, and the pathogenicity of the samples can be widely variable. Host susceptibility to these strains varies considerably between species. Experimental inoculations of P. multocida in mice and birds are commonly used to evaluate the pathogenicity of the different strains, but the results are generally subjective and difficult to evaluate them. The aim of this work was to establish a new methodology to classify the pathogenicity of a specific strain, through the formulation of a standard score. To determine this score, we used 97 samples of P. multocida, from fowl cholera in birds and rhinitis in swine. One-hundred microliters (105 CFU) of bacterial culture containing 106 CFU/mL of each P. multocida isolate were inoculated, by intraperitoneal route, in 10 one-day-old chicks. Besides mortality caused by the inoculation, time of death and gross lesions were also evaluated. Significant differences were observed between avian and swine isolates in relation to pathogenicity scores. The number of lesions and the percentage of bacteria recovered from the inoculated animals also varied according to the origin of the isolate. From the observed scores, the isolates were distributed into three pathogenicity classes: high, medium and low. The pathogenicity score developed here allows the measurement and classification of the pathogenicity of P. multocida isolates and can be an alternative to subjective current models of pathogenicity screening used so far.

Um total de 123 isolados de Pasteurella multocida provenientes de suínos foi avaliado através da PCR para pesquisa de genes codificadores de capsula, toxina dermonecrótica e outros fatores de virulência. As amostras foram caracterizadas como tipo capsular A (78,8 %) e tipo capsular D (21%). Nenhum dos isolados foi positivo para presença de toxina dermonecrótica. Os genes codificadores dos fatores de virulência pesquisados foram observados nas freqüências de 93,5 % para nanB, 92,7% para psl, 91,9% para oma87 e nanH, 87,8% para sodA,87% para hghA,83,7% para ompH, 82,9% para sodC, 79,7% para ptfA e exbBD tonB,73,2% para hgbB, 14,6% para pfhA e 4,9% para tbpA. Estes achados são compatíveis com o descrito na literatura internacional.
A total of 123 Pasteurella multocida strains from swine was evaluated through PCR to detect capsular, dermonecrotic toxin codifying genes and others virulence factors. The strains were identified as capsular type A (78.8 %) and capsular type D (21%). None of isolates were positive to dermonecrotic toxin gene. The virulence factors genes were detected in the following frequency: 93.5 % to nanB, 92.7% to psl, 91.9% to oma87 and nanH, 87.8% to sodA, 87% to hghA, 83.7% to ompH, 82.9% to sodC, 79.7% to ptfA and exbBD tonB, 73.2% to hgbB, 14.6% to pfhA and 4.9% to tbpA. These findings were in accordance with international literature.

Pasteurella multocida é o agente causador de muitas doenças de importância econômica em medicina veterinária e tem sido descrita como um agente de alto potencial zoónotico. No Brasil, ao contrário do que se observa na literatura internacional, informações sobre a frequência deste agente em animais de companhia como cães, gatos e coelhos são inexistentes. O presente estudo teve como proposta isolar cepas de Pasteurella multocida a partir de cães, gatos e coelhos, avaliar a freqüência deste agente nestas espécies animais, caracterizar os isolados de acordo com o tipo capsular e genes codificadores de fatores de virulência através da reação em cadeia pela polimerase, avaliar o perfil de resistência a antimicrobianos dos isolados obtidos e caracterizar as amostras através da PFGE. Foram examinados 640 animais, sendo 191 gatos, 309 cães e 140 coelhos. Dentre os animais examinados 8,1% foram positivos para o isolamento de P. multocida, sendo 10,4% dos gatos, 0,9% dos cães e 20,7% dos coelhos avaliados. Dentre as 93 cepas selecionadas 22,5% foram sensíveis a todas as drogas testadas, 77,4% das cepas foram resistentes a pelo menos uma droga utilizada e 5,3% foram apresentavam resistência a três ou mais drogas. Através da PFGE foram observados 39 pulsotipos com índice discriminatório igual a 0,97. Todos os genes codificadores de fatores de virulência foram detectados em pelo menos um isolado, sendo que nenhum destes esteve presente em 100% das cepas avaliadas.
Pasteurella multocida is the causative agent of many diseases of economic importance in veterinary medicine and has been described as an agent a high zoonotic potential. In Brazil, contrary to what is observed in the international literature, information about the frequency of this agent in animals such as dogs, cats and rabbits are nonexistent. This study proposes isolate strains of Pasteurella multocida from dogs, cats and rabbits, evaluate the frequency of this agent in these animal species, characterize the isolates according to the capsular type, and genes encoding virulence factors through the reaction polymerase chain, evaluate the antimicrobial resistance profiles of isolates and characterize the samples by PFGE. We examined 640 animals, 191 cats, 309 dogs and 140 rabbits. Among the animals examined were positive 8.1% for the isolation of P. multocida, and 10.4% of cats, dogs 0.9% and 20.7% of the rabbits studied. Among the 93 selected strains 22.5% were susceptible to all drugs tested, 77.4% of strains were resistant to at least one drug were used and 5.3% showed resistance to three or more drugs. Through PFGE were observed 39 pulsotipos discriminatory index equal to 0.97. All genes encoding virulence factors were detected in at least one isolated, none of which was present in 100% of the strains evaluated.

Pasteurella multocida is a causative agent of many diseases in a broad range of hosts. It is particularly noted in pigs as a cause of pneumonia and atrophic rhinitis. As treatment and control of P. multocida infections has relied heavily on antimicrobials, surveillance of antimicrobial resistance is necessary to ensure that treatment remains effective. The aim of this study was to investigate the antimicrobial resistance (AMR) of P. multocida isolates from Australian pigs, the potential risk of AMR genes spreading from other Gram-negative bacteria and the possibility of using bacteriophages as an alternative treatment to P. multocida infections. Antimicrobial susceptibility testing was performed on 273 P. multocida isolates collected from pig farms across Australia between 2014 and 2019. Resistance to tetracycline (22.7%), chlortetracycline (22%), florfenicol (0.7%) and ampicillin (0.4%) was identified. Examination of the transferability of AMR genes from ceftriaxone and ampicillin resistant E. coli isolates to P. multocida through plasmidmediated conjugation revealed that AMR genes were not stably transferred from E. coli to P. multocida. Efforts to isolate bacteriophages with lytic activity against P. multocida from environmental samples were not successful, however the addition of mitomycin C to P. multocida strains resulted in lysis of cells and visible clearing of the bacterial culture for 5 of 7 isolates, indicating prophages were induced. This study demonstrated that P. multocida infections in Australian swine can still be successfully treated with antimicrobials and that the risk of acquiring AMR genes from other highly resistant Gramnegatives is low. Despite low frequency of resistance to tested antimicrobials on-going surveillance and reducing antimicrobial usage should be a priority as P. multocida outbreaks can have significant impact on economic costs and animal welfare. Failure to isolate lytic bacteriophages from environmental sources indicates that phage therapy would be reliant on isolation of phage stocks from sources samples that would carry P. multocida specific phages such as nasopharyngeal wash samples from pigs.

Pasteurella multocida é um dos principais patógenos envolvidos nas broncopneumonias infecciosas em suínos. Apesar de ser considerada agente secundário à pneumonia enzoótica causada pelo Mycoplasma hyopneumoniae e por agentes virais como o vírus da influenza suína, há evidências do seu envolvimento como agente primário. Neste contexto, o primeiro estudo desenvolvido teve como objetivo desenvolver um método de reprodução experimental de pneumonia por P. multocida A cepa 11246 em suínos infectados com diferentes concentrações de inóculo. Um segundo estudo foi desenvolvido com o objetivo de demonstrar diferenças fenotípicas, moleculares e patogênicas entre cepas de P. multocida A isoladas de casos clínicos de pneumonia em granjas comerciais de suínos de vários estados brasileiros. No primeiro experimento os suínos foram desafiados por gotejamento intranasal lento com inóculo de diferentes concentrações de P. multocida A cepa 11246 [Grupo (G1): 108 Unidades Formadoras de Colônias (UFC)/ml; G2: 107 UFC/ml; G3: 106 UFC/ml e G4: 105 UFC/ml]. Foram utilizados dois suínos por grupo com aproximadamente 100 dias de idade. Neste, todas as concentrações de inóculo demonstraram a capacidade da bactéria em causar doença respiratória grave e septicemia nos animais inoculados. Utilizando-se a mesma metodologia de desafio, com inóculo de 107 UFC/ml, o segundo estudo, ao desafiar 64 suínos igualmente distribuídos em oito grupos (G1 a G8) com oito diferentes cepas de P. multocida A (uma cepa por grupo), os resultados demonstraram a presença de cepas muito patogênicas (G1-11246, G2-11229, G3-16614 e G7-17044); pouco patogênicas (G4-16618 e G5-16972); e apatogênicas (G6-17034 e G8-17078), de acordo com a gravidade das alterações clinico-patológicas desenvolvidas. Na avaliação patológica dos animais desafiados, observaram-se três padrões de lesões distintas, associadas ou não entre si: 1. broncopneumonia fibrinonecrótica cranioventral com pleurite fibrinosa (G1, G3, G7); 2. pleurite difusa uni ou bilateral, associada ou não com pericardite e peritonite (G3, G5, G7) e; 3. pleuropneumonia necrossupurativa focal, geralmente no lobo cardíaco (G1, G2; G3, G4, G7). Na análise genotípica, nos padrões de PFGE obtidos após a macro-restrição com a enzima ApaI, as cepas patogênicas (Nos 11246, 11229, 16614, 16618, 16972 e 17044) foram classificadas no mesmo grupo, com homologia variando de 67,3 a 100%, diferenciando-se das cepas apatogências (Nos 17034 e 17078), que pertenceram a outro grupo, com homologia de apenas 52,7% com as demais amostras. Coletivamente, os resultados demonstraram padrões distintos de patogenicidade de diferentes cepas de P. multocida, os quais podem estar associados à características genéticas das cepas. Adicionalmente o estudo demonstrou a atuação primária de algumas cepas de P. multocida em pneumonias, pleurites e septicemias em suínos.
Pasteurella multocida is one of the main pathogens involved in infectious bronchopneumonia in swine. Although considered a secondary agent to the enzootic pneumonia caused by Mycoplasma hyopneumoniae and viral agents as the swine influenza, there are evidences related to its involvement as a primary agent. In this context, the first study undertaken aimed at developing an experimental reproduction method of pneumonia caused by P. multocida A Strain 11246 in swine infected with different inoculum concentrations. A second study was conducted aiming demonstrating phenotypic, molecular and pathogenic differences between the strains of P. multocida A isolated from clinical cases of pneumonia in commercial swine farms in several Brazilian states. In the first experiment, swine were challenged by slow intranasal drip with different inoculum concentrations of P. multocida A strain 11246 [Group (G1): 108 Colony Forming Units (CFU)/ml; G2: 107 CFU/ml; G3: 106 CFU/ml and G4: 105 CFU/ml]. Two swine per group with approximately 100 days of age were used. In these animals all inoculum concentrations demonstrated the bacteria capability to cause severe respiratory disease and septicemia in the inoculated animals. Using the same challenge methodology inoculating 107 CFU/ml at the second study when challenging 64 swine equally distributed into eight groups (G1 to G8) with eight different strains of P. multocida A (one strain per group) results showed the presence of highly pathogenic strains (G1-11246, G2- 11229, G3-16614 e G7-17044); less pathogenic (G4-16618 e G5-16972); and apathogenic (G6-17034 e G8-17078), according to the severity of the clinical and pathological alterations developed. In the pathologic evaluation of challenged animals, we observed three distinct patterns of injuries associated or not with each other: 1. Cranioventral fibrinonecrotic bronchopneumonia with fibrinous pleuritis (G1, G3, G7); 2. Difuse uni or bilateral pleuritis pleuritis, associated or not with pericarditis and peritonitis (G3, G5, G7) and; 3. Necrosuppurative focal pleuropneumonia, generally in the cardiac lobe (G1, G2, G3, G4, G7). In genotypic analysis, the PFGE patterns obtained after the macro-restriction with ApaI enzyme, the pathogenic strains (# 11246, 11229, 16614, 16618, 16972 and 17044) were classified in the same group, with homology ranging from 67.3 to 100%, differing from the apathogenic strains (# 17034 and 17078), which belonged to another group, with only 52.7% homology with the other samples. Collectively, the results showed distinct patterns in different pathogenic strains of P. multocida, which may be associated with the genetic features of the strains. Additionally, the reasearch demonstrated the primary role of some strains of P. multocida in pneumonia, pleuritis and septicemia in swine.

Los objetivos de la presente tesis doctoral han sido la caracterización de los mecanismos de captación de hierro de Pasteurella multocida, con el fin de contribuir al diseño de una vacuna contra este patógeno basada en la sobreexpresión de proteínas de membrana externa, receptoras de hemoglobina y/o hemina, o en la expresión constitutiva de las proteínas de membrana reguladas por hierro (IROMPs).
Se ha identificado el gen fur de P. multocida y se ha estudiado el perfil electroforético de las proteínas de membrana externa de dicho patógeno, descubriéndose un control de la expresión de una proteína mayoritaria de membrana (OmpH) por parte de la proteína Fur. Se ha construido un mutante fur y se ha demostrado que la mutación de este gen no presenta ningún efecto sobre la virulencia de la cepa.
Así mismo, se ha caracterizado la región cromosómica de P. multocida que contiene los genes exbB, exbD y tonB, detectándose la existencia de promotores internos en dicha región y por lo tanto la expresión independiente de cada uno de los tres genes del sistema. Se ha estudiado la regulación de la expresión de todos ellos, demostrándose que pertenecen al regulón Fur, y se han construido mutantes en los genes exbB, exbD y tonB. Se ha calculado la dosis letal 50 (DL50) de cada una de las cepas construidas, determinándose que cada uno de estos genes es imprescindible para el desarrollo normal de una infección por P. multocida, ya que en todos los casos se obtuvo un incremento de la DL50 superior a los tres órdenes de magnitud con respecto la cepa salvaje.
Por otro lado, se ha analizado el genoma de P. multocida Pm70 y se han estudiado 9 proteínas que, por similitud con los receptores descritos en el banco de datos, podrían actuar como receptores de hemoglobina y/o hemina. Así se ha podido determinar que algunas de ellas son capaces de unir hemoglobina y hemina (PM0040, PM0236, PM0300, PM0741, PM1081 y PM1428), mientras que otras tan sólo interaccionan con la hemoglobina (PM0576) o la hemina (PM1282), y que otra (PM1078) no reconoce ninguna de estas fuentes de hierro.
De entre todas estas proteínas se ha elegido la PM0300, que se ha denominado HgbA, para realizar un estudio más detallado de este tipo de receptores de membrana. Así, se ha demostrado que el gen hgbA forma una unidad transcripcional con los genes PM0298 y PM0299. Estos genes presentan similitud con hugX y hugZ de Plesiomonas shigelloides respectivamente, y están implicados en procesos de detoxificación de hierro. Se ha demostrado que, en P. multocida, las proteínas PM0298 y PM0299 son esenciales para la viabilidad de la cepa. Por otro lado, se ha determinado que el gen hgbA se expresa en las primeras dos horas de la infección y que, in vitro, su expresión se induce en ausencia de hierro. Además, se ha construido un mutante deficiente para esta proteína y se ha comprobado que no presenta disminuida ni su capacidad de unir hemoglobina in vitro ni su virulencia. Por otro lado, se ha comprobado la distribución prácticamente universal de este gen en cepas de P. multocida de distintos serotipos y orígenes animales, hecho que propone a esta proteína como candidata para el desarrollo de un método de identificación rápido y específico de este microorganismo.
Por último, se ha analizado la capacidad inmunogénica y protectora de los receptores de hemina y/o hemoglobina. Así, se ha demostrado que por lo menos tres de estas proteínas (PM0236, PM0741 y HgbA) son inmunogénicas. Sin embargo, la inoculación en ratones de las proteínas HgbA y PM0741, ya sea de forma individual o conjunta, no induce protección ante un enfrentamiento homólogo.
In the present work, the Pasteurella multocida iron uptake mechanisms have been characterised. These results could be use to the obtention of one vaccine against this pathogen.
The P. multocida fur gene has been identified and it has been demonstrated that the porine OmpH is under the control of the Fur protein. On the other hand, a P. multocida Fur mutant was constructed and its LD50 was calculated demonstrating that a mutation in this gene has no effect in the virulence of the strain.
Moreover, the region which contains P. multocida exbB, exbD and tonB genes has been characterised. The results obtained demonstrate that these three genes do not constitute a single transcriptional unit, but all these genes possess its own promoter which is under the Fur-Fe(II) regulation. It has been obtained a ExbB, ExbD and TonB mutants and these mutants present a decrease in their virulence to increase their LD50 by more than three orders of magnitude when were inoculated via intraperitoneal. These data demonstrate that exbB, exbD and tonB genes are essential to P. multocida infectious process.
Furthermore, the P. multocida Pm70 genome was analysed and nine putative haemin and haemoglobin-binding proteins have been identified by similarity with the same molecules of other bacterial pathogens. Quantitative binding assays have demonstrated that PM0040, PM0236, PM0300, PM0741, PM1081 and PM1428 bind both haemin and haemoglobin, whereas PM0576 and PM1282 only bind either haemoglobin or haemin, respectively. PM1078, despite presents similarity with a Yersinia enterocolitica haemin receptor, does not recognize none of these iron sources.
PM0300 was named hgbA and it has been demonstrate that this gene and the PM0298 and PM0299 ORFs, which present similarity with hugX and hugZ of Plesiomonas shigelloides, respectively, constitute a single transcriptional unit. PM0298 and PM0299 are essential to the viability of P. multocida cells. The hgbA gene is expressed in vivo within the two first hours post-inoculation, and in vitro is repressed by iron. Moreover, a HgbA mutant binds haemoglobin to the same extent as the wild-type strain and, in agreement with this, virulence of the P. multocida hgbA cells was no affected. On the other hand, the hgbA gene is present in almost all the strains of P. multocida analysed, independently of their serotype or their animal source. This result propose hgbA as a putative candidate to develop a fast and specific method to identify this pathogen.
Finally, the immumogenicity and the ability to induce protection of all these haemin and haemoglobin receptors were analysed. The results obtained have demonstrated that, despite PM0236, PM0741 and HgbA are immunogens, inoculation of mice with any single one of these binding proteins alone is not protective against P. multocida infection.

The heterotrimeric G-proteins are critically important intracellular signalling molecules that regulate fundamental processes in cellular homeostasis. A unique bacterial toxin, Pasteurella multocida toxin (PMT) modifies 3 of the 4 families of G-proteins (Gq, Gi, and G12) by deamidation which leads to a plethora of downstream signalling. PMT induces drastic morphological changes such as loss of adherence to the matrix, foci and stress fibre formation in Swiss 3T3 and Vero cells. PMT is mitogenic for many cell types but not all, and the work reported in this thesis aims to compare two cell lines that respond differently to PMT. Swiss 3T3 and Vero cells were treated with different concentrations of PMT to determine the effects on cell proliferation, cytoskeletal reorganisation and cell death. PMT induced prominent cytoskeletal changes, mitogenic and anti-cell death responses in Swiss 3T3 cells while delayed cytoskeletal changes with no evidence of mitogenicity and cell death were observed in Vero cells. PMT modified G-proteins at different times in Swiss 3T3 and Vero cells. The PMT-induced anti-cell death response in Swiss 3T3 cells was dose-dependent while the delayed cytoskeletal response in Vero cells was linked to the late PMT-mediated G12 activation. The amino acid sequence of Gα12 differed in the two cell types – the G12 subunit in Vero cells is missing N-terminal cysteine residue, which may have contributed to the differences. Gq/11 signalling is active and sustained in Swiss 3T3 cells, but not in Vero cells. Gβγ may have inhibited adenylyl cyclase activity so it is unknown whether Gi signalling is active and sustained over the 3-day period as the forskolin-stimulated cAMP level decreased in both serum-starved untreated and PMT-treated cell lines. In summary, I have identified changes in both the primary effect of PMT on the two cell types and also on downstream signalling that is likely to reflect the differential cell response.

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.

Caracteriza molecularmente 24 aislados de P. multocida provenientes de alpacas con neumonía, identificados por pruebas bioquímicas y mediante la prueba de PCR a travez de la amplificando del gen kmt1 presente solo en esta especie. Posteriormente se realizó la tipificación capsular mediante la técnica de PCR múltiple obteniéndose que todos los aislados pertenecen al tipo capsular A. La tipificación en base al antígeno LPS, el cual permite la clasificación de P. multocida en 16 serovares agrupados en 8 genotipos, resultó en que los aislados de este estudio pertenecieron al genotipo LPS L6, correspondiente a los serovares 10, 11, 12 y 15. Además, se analizó la presencia de 4 genes codificantes de factores de virulencia (toxA, tbpA, pfhA y hgbB), detectándose en todos los aislados los genes toxA (100%) y tbpA (100%). Para evaluar la diversidad entre los aislados, se emplearon las técnicas BOX-PCR y ERIC-PCR, los cuales fueron analizados por el programa NTSYSpc 2.10 para el desarrollo de los dendogramas mostrando baja diversad entre los aislados de alpaca, corroborado por el agrupamiento de 23/24 aislados en un único cluster.
Tesis

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For a long time, Pasterella multocida has been considered only as a secondary agent in pneumonias in swine. However, studies developed in Brazil identified different isolates of P. multocida able to operate as primary disease agent in swine. The objective of his project was to study protection aspects of a vaccine with pathogenic samples of P. multocida in experimental model with high sanity status pigs. In addition, it aims to describe the expression of immune response in swine induced by P. multocida infection, and determinate the prevalence of P. multocida in tonsils of vaccinated and not vaccinated pigs through immunohistochemistry. The study was divided into three experiments: Immunization of mice with vaccines prepared with different adjuvants , Test of two adjuvants used in vaccine with a P. multocida homologous strain in swine and Experimental challenge in swine with a P. multocida heterologous strain to the vaccine . Four adjuvants were tested in mice: Al(OH)3, ISA 206, Montanide GEL 01 and ISA 760. Two of them were selected for testing in swine: Al(OH)3 which showed better results in mice-, and ISA 760 for being an oily adjuvant. At the second experiment, although two vaccines have been effective in the disease protection, the one with the adjuvant Al(OH)3 was selected, because showed lower vaccine reaction. Eighteen pigs were distributed into three groups for the specific test of the vaccine for pathogenic samples of P. multocida. Group 1 (G1) vaccine with Al(OH)3; group 2 (G2) infection control; and group 3 (G3) negative control. After the experiment, the animals were euthanized, and samples were collected in ice - for microbiological cultivation and relative quantification of cytokines gene expression (IL1 β, IL2, TNFα) and TLR4 , and in formaldehyde, for routine histopathology and immunohistochemistry. Results showed that the vaccine prototype with adjuvant Al(OH)3 was efficient in controlling pulmonary pasteurellosis. The immune response was mediated by TLR4, with increased expression of TNFα. IL-1β and IL2 were also expressed, however, there was no significant difference between the groups. The immunostaining of P. multocida in tonsil was positive in all pigs challenged with the bacteria
Por muito tempo, Pasteurella multocida foi considerada apenas como agente secundário nas pneumonias em suínos, todavia, estudos realizados no Brasil identificaram diferentes isolados de P. multocida capazes de atuar como agente primário de doença em suínos. Esse projeto teve como objetivo estudar aspectos de proteção de uma vacina com amostras patogênicas da P. multocida em modelo experimental com suínos de alto status sanitário . E de forma paralela descrever expressão da resposta imune dos suínos induzida pela infecção da P. multocida e determinar a prevalência dessa bactéria em tonsilas de suínos vacinados e não vacinados através de imunohistoquímica. O trabalho foi dividido em três experimentos: Imunização de camundongos com vacinas preparadas com diferentes adjuvantes ; Teste de dois adjuvantes utilizados na vacinação de suínos frente ao desafio com uma cepa homóloga de P. multocida ; e Desafio experimental em suínos com uma cepa de P. multocida heteróloga à da vacina . Em camundongo foram testados quatro adjuvantes: Al(OH)3, ISA 206, Montanide GEL 01 e ISA 760, dos quais, foram selecionados dois para teste em suínos: Al(OH)3, que apresentou melhor resultado nos camundongos e ISA 760, por ser um adjuvante oleoso. Embora as duas vacinas tenham sido efetivas na proteção da doença, no segundo experimento, selecionou-se àquela com adjuvante Al(OH)3, que apresentou menor reação vacinal. No terceiro experimento realizado em suínos, foram utilizados 18 suínos em três grupos: grupo 1 (G1) - vacina com Al(OH)3, e grupo 2 (G2) controle de infeção e grupo 3 (G3) controle negativo. Após o experimento os animais foram eutanasiados para avaliação patológica, coleta de amostras em gelo para cultivo microbiológico e quantificação relativa da expressão gênica das citocinas (IL1 β, IL2, TNFα) e TLR4, para histopatologia de rotina e imunohistoquimica. Os resultados demostraram que o protótipo de vacina com adjuvante Al(OH)3 foi eficiente no controle de pasteurelose pulmonar. O perfil da expressão da resposta imune foi mediado por TLR4, com aumento da expressão de TNFα. Também foram expressos IL-1β e IL2, porém sem diferença significativa entre os grupos. A imunomarcação de P. multocida na tonsila foi positiva em todos os suínos desafiados com a bactéria, independente de serem ou não vacinados

Une banque genomique de p. Multocida a ete realisee et criblee avec un serum dirige contre une proteine majoritaire de la membrane externe de p. Multocida, la porine h de 37 kda. Un clone (e. Coli puca) a ete selectionne pour ses proprietes de marquage par les anticorps. E. Coli puca contient un fragment d'adn genomique de p. Multocida de 2,9 kpb. Il exprime un peptide de 25 kda (p25) localise dans le periplasme et reconnu par le serum anti-porine h. Des anticorps polyspecifiques diriges contre les composants des enveloppes de p. Multocida ont permis de montrer que l'insert exprime aussi un peptide de 28 kda (p28). Une construction genetique (e. Coli pucb) a permis de surproduire les antigenes p25 (6% des proteines de la fraction soluble) et p28 (20% des proteines membranaires). Une carte de restriction ainsi que des sous-clones ont ete realises pour localiser les genes correspondant a p25 et p28 et permettre le sequencage de l'adn et la construction de sondes nucleotidiques. Des experiences d'hybridation adn-adn ont montre que l'insert est un fragment d'adn continu de p. Multocida en exemplaire unique dans le genome. Le sequencage de l'insert de 2,9 kpb decrit dans cette these montre l'existence de 3 phases ouvertes de lecture codant pour des peptides de 42 kda, 21 kda et 36 kda. La comparaison de la sequence d'adn avec les sequences n-terminales des peptides p25 et p28 montre que p28 est le precurseur de p25 et que le gene se situe au niveau de la phase ouverte correspondant au peptide de 21 kda. Cette sequence d'adn est homologue des genes omph de s. Typhimurium et hlpa (=skp) de e. Coli. D'autre part, la phase ouverte de 36 kda possede des homologies importantes avec les genes ssc de s. Typhimurium et fira d'e. Coli qui sont impliques dans la transcription. A partir de plusieurs sondes d'adn marquees a la digoxigenine, nous avons etudie le polymorphisme de restriction sur 9 souches de p. Multocida. Les resultats nous ont permis de classer les souches de pasteurelles en 3 groupes

Apesar de ser uma bactéria que compõe a microbiota respiratória, sob algumas circunstâncias pode manifestar-se como um agente patogênico primário ou secundário, causando doença em aves e outros animais. Como agente primário, P. multocida leva a grandes perdas econômicas, causando cólera em aves, rinite atrófica em suínos e septicemia hemorrágica em bovinos e bubalinos. P. multocida é uma espécie heterogênea e a patogenicidade dos isolados pode ser amplamente variável. A suscetibilidade do hospedeiro a essas cepas varia consideravelmente entre espécies. Inoculações experimentais em camundongos e aves são comumente usadas para avaliar a patogenicidade de diferentes cepas, mas os resultados são geralmente subjetivos e pouco mensuráveis. O objetivo deste trabalho foi estabelecer uma nova metodologia para classificar a patogenicidade de cepas de P. multocida, através da formulação de um índice padrão. Para determinar esse índice, foram selecionadas 97 amostras de P. multocida, isoladas de cólera em aves e rinite em suínos. Cem microlitros de uma cultura bacteriana contendo 103 UFC/mL de cada isolado de P. multocida, foram inoculados pela cavidade córioalantoide, em 3 ovos embrionados. Além da mortalidade causada pela infecção, o tempo de morte e as lesões macroscópicas foram avaliados. Diferenças significativas foram observadas entre isolados de aves e suínos em relação aos índices de patogenicidade. O número de lesões e o percentual de bactérias recuperadas dos embriões inoculados também variaram de acordo com a origem do isolado. A partir dos índices observados, os isolados foram distribuídos em três classes de patogenicidade: alta, média e baixa. A avaliação dos diferentes índices de patogenicidade, estudados neste trabalho, permitem o estabelecimento de novos modelos de avaliação de patogenicidade de isolados de P. multocida e podem ser uma alternativa aos modelos subjetivos até então utilizados. A comparação dos índices de patogenicidade obtidos nos diferentes modelos analisados nos permite afirmar que a variação esperada para os diferentes modelos não pode ser observada. A análise dos diferentes perfis genéticos obtidos para as cepas de origem aviária, a partir da clivagem do gene ompH e da técnica de PCR-RFLP também não revelou diferença estatística entre os perfis obtidos e suas respectivas patogenicidades obtidas no modelo experimental de camundongos.
Although it is a bacterium that makes up the respiratory microbiota, under some circumstances it may manifest itself as a primary or secondary pathogen, causing disease in birds and other animals. As a primary agent, P. multocida leads to severe economic losses, causing cholera in birds, atrophic rhinitis in pigs and hemorrhagic septicemia in cattle and buffaloes. P. multocida is a heterogeneous species and the pathogenicity of the isolates can be widely variable. The susceptibility of the host to these strains varies considerably between species. Experimental inoculations in mice and birds are commonly used to evaluate the pathogenicity of different strains, but the results are generally subjective and poorly measurable. The objective of this work was to establish a new methodology to classify the pathogenicity of P. multocida strains by formulating a standard index. To determine this index, 97 samples of P. multocida, isolated from cholera in birds and rhinitis in swine, were selected. One hundred microliters of a bacterial culture containing 103 CFU / mL of each P. multocida isolate were inoculated by the chorioallantoic cavity in 3 embryonated eggs. In addition to infection mortality, time of death and macroscopic lesions were assessed. Significant differences were observed between isolates of birds and pigs in relation to pathogenicity indexes. The number of lesions and the percentage of bacteria recovered from the inoculated embryos also varied according to the origin of the isolate. From the observed indices, the isolates were distributed in three pathogenicity classes: high, medium and low. The evaluation of the different pathogenicity indexes, studied in this work, allows the establishment of new pathogenicity evaluation models of P. multocida isolates and may be an alternative to the subjective models previously used. The comparison of the pathogenicity indices obtained in the different models analyzed allows us to state that the variation expected for the different models can not be observed. The analysis of the different genetic profiles obtained for the strains of avian origin, from the cleavage of the ompH gene and the PCR-RFLP technique also did not reveal statistical difference between the profiles obtained and their respective pathogenicities obtained in the experimental model of mice. Key words: Pasteurella multocida, pathogenicity index, embryonated eggs, mice, chicks.

Cette these est consacree a la purification et a la caracterisation de la proteine majoritaire (porine h) de la membrane externe de pasteurella multocida, une eubacterie gram-negative responsable de maladies respiratoires chez le porc. La purification de cette proteine sous forme denaturee et monomerique (37 kda) et sous forme native et trimerique a permis sa caracterisation structurale et fonctionnelle. Les experiences sur films noirs montrent que la proteine h est effectivement une porine formant des canaux transmembranaires de 0,9 nm de diametre. Les proprietes biochimiques de la proteine h sont typiques des porines bacteriennes. Les homotrimeres de proteine h sont totalement dissocies par le sds en monomeres apres chauffage a 100c. Les analyses en dichroisme circulaire montrent que la porine h native possede une conformation riche en feuillets beta. Les analyses d'image de reseau cristallin bidimensionnel de proteine h, apres contraste negatif en microscopie electronique, revelent une structure trimerique de 7,7 nm de diametre avec 3 canaux par trimere. Les comparaisons quantitatives des compositions en aminoacides montrent que la proteine h est apparentee a la porine p2 d'haemophilus influenzae (une bacterie appartenant a la famille des pasteurellaceae) et aux porines des neisseria. Les comparaisons n-terminales confirment que la porine h de p. Multocida est apparentee a la porine p2 et aux porines non specifiques des enterobacteries. La proteine h est antigeniquement conservee chez les souches de p. Multocida. Un potentiel vaccinal effectif mais modere de la porine h, presentee sous forme d'iscoms, a ete obtenu sur souris

Haemorrhagic septicemia (HS), caused by the Gram-negative bacterium Pasteurella multocida serotype B:2, is an economically important disease responsible for morbidity and mortality of bovines, especially buffaloes, in countries of South or Southeast Asia and Africa. A feature of this disease is the rapid spread of infecting bacteria from the respiratory tract to the blood and lymph to cause a fatal septicemia. To pass into the blood stream, the bacteria must migrate through the epithelial layer into the pulmonary interstitium. Avian serogroup A strains of P. multocida have been reported to invade cultured mammalian cells, but the behaviour of other of serogroups has not been reported. The main object of the work was to confirm that HS strains of P. multocida B:2 have the capacity to invade and survive within cultured mammalian cells, such as J774.2 cells (mouse macrophage-like cell lines) and BL-3 cells (bovine lymphoma cell line). Invasion, defined as adhesion to, followed by uptake by, or entry into, J774.2 macrophage cells or BL-3 cells was determined by: (I) counting of viable intracellular bacteria after killing extracellular bacteria with polymyxin and gentamicin, (II) Transmission electronic microscopy. Comparison of the invasiveness of a B:2 HS strain and its aroA derivative JRMT12 with that of P. multocida A:3 and E. coli XL1-Blue, showed that both P. multocida B:2 strains invaded both types of mammalian cells more readily than P. multocida A:3 and that E. coli XL1-Blue was essentially non-invasive. Both strains of P. multocida B:2 could survive within J774.2 macrophage and BL-3 cells for at least 2 h. A longer-term survival experiment (up to 6 h incubation) indicated that the numbers of intracellular bacteria declined between 4 to 6 h post-infection. It was shown by TEM that a significant proportion of the P. multocida B:2 bacteria were found within vacuoles in the cytoplasm of the mammalian cells with some free in the cytoplasm. A much reduced invasion capacity of P. multocida A:3 and E. coli XL1-Blue was detected. Different effects on the appearance and viability of J774.2 and BL-3 cells were observed by the trypan blue method and TEM when exposed to the P. multocida B:2 strains. Evaluation of cytotoxicity of P. multocida B:2 stains with J774.2 cells by the MTT assay produced unsatisfactory results.

The project was concerned with the construction of defined attenuated derivatives of P. multocida serotype B:2 strains, causative agents of haemorrhagic septicaemia, and attempts were made to construct defined mutations in genes such as aroA, cya, and galE loci that have been used to induce attenuation in other bacterial strains. Mutants defective in the aroA gene were constructed by allelic exchange of the locus in the chromosome of the wild-type strains with a cloned aroA gene interrupted with a cassette encoding kanamycin resistance (KmR). The aroA defective strains were confirmed by PCR, Southern blotting, lack of growth on minimal medium and by enzyme assay. KmR inactivated aroA mutants JRMT1 and JRMT2 strains derived from P. multocida 85020 and Quetta strains, respectively, were highly attenuated in a mouse model, with an LD50 108 C.F.U./mouse after injection intraperitoneally (i.p.). In contrast, the wild-type strains had LD50 <50 C.F.U./mouse by this route. Vaccination once by the i.p. route or twice by the i.n. route with these aroA mutants gave complete protection to the mice against subsequent challenge i.p. with 10,000 LD50 of the homologous wild-type strain or 1000 LD50 of the heterologous wild-type strain. Vaccination with these by the s.c. route was not protective. When high doses of the attenuated strains were inoculated by the i.p. or i.n. routes, there was some spread to the internal organs but the organisms were cleared by 24 and 72 hrs respectively. In contrast, the wild-type parent strains spread rapidly and multiplied in high numbers and killed the mice by 24 and 96 hrs respectively.

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.

Studies were conducted in order to investigate the role of Pasteurella multocida in porcine enzootic pneumonia (PEP). The prevalence and extent of PEP and pleurisy lesions were determined in several classes of pigs from PEP-affected and specific pathogen-free (SPF) herds. The association between selected environmental/managerial risk factors (class, season, climatic zone, herd-size, sex and sow parity) and the outcomes of PEP and pleurisy was investigated. The correlation between PEP and pleurisy scores at slaughter and the growth rate of pigs was determined. The recovery of P.multocida and other non-fastidious organisms from pig lungs at slaughter was compared. P.multocida was most commonly isolated. The herd prevalence of P.multocida pulmonary infection was 100%, which was higher than that of any other organism. Only P.multocida was associated with the presence and extent of PEP lesions. The recovery of other organisms was markedly reduced when bacterial contamination of the lungs during slaughter was avoided by scalding carcasses with steam rather than by immersion in water. The chronological pattern of the prevalence of P.multocida nasal infection between weaning and slaughter was investigated. The same selected environmental/managerial risk factors examined with respect to PEP and pleurisy were assessed with respect to association with P.multocida pulmonary infection. The correlation between P.multocida pulmonary infection and growth rate was determined. A subset of presumptive P.mu1!ocida isolates was subjected to capsular typing, somatic serotyping, testing for production of dermonecrotoxin, SDS-PAGE profiling of whole cell proteins and outer membrane proteins (OMPs), biochemical activity testing, subspeciation and multilocus enzyme electrophoresis (MEE). Most lung isolates were capsule type A whereas most nasal isolates belonged to capsule type D. Somatic serotypes 3 and 3,12 were most common. Several correlations were observed between the MEE type of isolates and their capsule type, somatic serotype, biochemical activity and subspecies classification. The outer membrane (OM) profile of a reference PEP strain of P.multocida was examined under a variety of conditions using SDS-PAGE and immunoblotting. There was increased expression of several high molecular weight proteins in OM prepared from cells grown under iron-restricted conditions. Several individual bands in OM preparations were recognised on immunoblots by antisera made in pigs. The absence of B—mercaptoethanol from SDS-PAGE sample buffer had no effect on the OM profile. OM contained at least two heat modifiable proteins. Most OM bands were accessible to antibodies and enzymic digestion while in-situ on whole cells. The OM profile was compared with that of a number of aqueous extracts of whole cells. Several crude antigens were examined with respect to their suitability as ELISA antigens for use in the serodiagnosis of P.multocida infection in conventional pigs managed under field conditions. A higher antibody titre to each of the crude antigens was detected in sera from conventional rather than SPF pigs, however all crude antigens cross-reacted extensively with other members of the family Pasteurellaceae. A 29 kDa OMP, which was antigenically-related in many P.multocida isolates as determined by immunoblotting, was purified from OM by electroelution from SDS-PAGE gels. It's cross-reactivity, as assessed using ELISA and immunoblotting, was reduced in comparison with that of the crude antigens. Serial serum samples taken from conventional pigs between weaning and slaughter were tested using ELISA for antibodies to Mycoplasma hyopneumoniae, Mycoplasma hyorhinus and P.multocida (using the 29 kDa 0MP as antigen). Pigs began to seroconvert to P.multocida six weeks earlier than to Mhyopneumoniae. Nearly all pigs seroconverted to P.multocida during the period of observation, whereas only a proportion seroconverted to each of the two Mycoplasma pathogens. Only the Mhyopneumom'ae titre was positively associated with the presence and extent of PEP lesions. Mhyopneumoniae and particularly Mhyorhinus titres were positively associated with pleurisy. Only the P.multocida titre was negatively associated with growth rate. Pigs which were lung culture-positive for P.multocida but sero-negative for Mhyopneumoniae often had significant PEP lesions at slaughter and these were larger than those in pigs which were lung culture-negative for P.muItocida but sero-positive for Mhyopneumoniae. A mouse model of pneumonic pasteurellosis was established in which to evaluate the protective potential of OM antigens. Despite intratracheal inoculation, the resulting clinical syndrome was essentially septicaemic rather than pneumonic in nature. Bacterins and OM preparations induced strong immunity against high level homologous challenge, particularly when given intraperitoneally. Production of these vaccines from cells grown under ironrestn'cted conditions did not increase the level of immunity induced. The effect of a number of physical and chemical treatments on the immunogenicity of an OM preparation was investigated. Partial purification of individual OM antigens using mild SDS-PAGE resulted in some fractions which induced 80-100% protection against low level challenge, however it was not possible to correlate protection with the presence of particular bands in those fractions. No protection was associated with any fraction containing highly purified individual OM antigens. A cluster of very high molecular weight OM antigens partially purified using mild SDS-PAGE was moderately protective against high level challenge. The ability of various antigens to induce cross-protection against challenge with heterologous strains of P.multocida was investigated. in some fractions which induced 80-100% protection against low level challenge, however it was not possible to correlate protection with the presence of particular bands in those fractions. No protection was associated with any fraction containing highly purified individual OM antigens. A cluster of very high molecular weight OM antigens partially purified using mild SDS-PAGE was moderately protective against high level challenge. The ability of various antigens to induce cross-protection against challenge with heterologous strains of P.multocida was investigated.

Pasteurella multocida is an important multi-host animal and zoonotic pathogen that is capable of causing respiratory and multi-systemic diseases, bacteremia, and infections resulting from bite wounds. The glycosaminoglycan capsule (CPS) of P. multocida is an essential virulence factor, protecting the bacterium from host defenses. However, chronic infections such as bovine respiratory disease (BRD) and avian cholera may be associated with biofilm formation. Biofilm formation was inversely related to capsule production (determined by uronic acid and N-acetylglucosamine assays), and was confirmed with capsule-deficient mutants of mucoid strains. Capsule-deficient mutants formed biofilms with a larger biomass that was much thicker and smoother than encapsulated strains. Gas chromatography-mass spectrometry, nuclear magnetic resonance, and enzymatic digestion demonstrated that the matrix material of the biofilm was composed predominately of a glycogen exopolysaccharide (EPS). Therefore, CPS may interfere with biofilm formation by blocking adherence to a surface or by preventing the EPS matrix to encase large numbers of bacterial cells. Chemical mutagenesis was performed on P. multocida strain P1059, resulting in isolation of an acapsular mutant designated as P1059-R8. A uridyltransferase encoded by gene P1059_01979 was mutated in such a way that a polar amino acid was changed to a non-polar amino acid near the active site. The protein product of P1059_01979 is important for the biosynthesis of the CPS subunit N-acetylglucosamine. CPS quantification revealed that the subunit glucuronic acid was produced in equal concentrations to the parent, but the CPS subunit N-acetylglucosamine was not detected in the chemical mutant. Biofilm formation in the chemical mutant was significantly higher than in WT P1059 and the capsule-deficient mutant. We hypothesize that P1059_01979 is essential for CPS production in P. multocida serogroup A. Histophilus somni and Pasteurella multocida cause bovine respiratory disease (BRD) and systemic infections in cattle. Following respiratory infection of calves with H. somni, P. multocida is also often isolated from the lower respiratory tract. Because H. somni normally forms a biofilm during BRD, we suspected that P. multocida may co-exist with H. somni in a polymicrobial biofilm. Interactions between the two species in the biofilm were characterized and quantified by fluorescence in situ hybridization (FISH), and the biofilm matrix of each species examined by fluorescently-tagged lectins (FTL), confocal scanning laser microscopy of in vitro biofilms and bovine pulmonary tissue following dual H. somni and P. multocida infection. FISH and FTL were used to show that P. multocida and H. somni were evenly distributed in the in vitro biofilm, and both species contributed to the polymicrobial biofilm matrix. COMSTAT z-stack image analysis revealed that the average biomass and biofilm thickness of the individual and polymicrobial biofilms were greatest when both species were present. Encapsulated P. multocida isolates not capable of forming a biofilm still formed a polymicrobial biofilm with H. somni, but only the EPS of H. somni could be detected by FTL staining of bovine tissues from which both species were isolated. Bacteria within a biofilm are more quiescent than during planktonic growth and induce less of an inflammatory response, indicating encapsulated P. multocida may take advantage of the H. somni biofilm to persist in the host during less severe, but more chronic, BRD. These results may have important implications for the management of BRD. Acute avian cholera is associated with encapsulated P. multocida, while chronic and asymptomatic cases of avian cholera are associated with acapsular P. multocida isolates. We hypothesize that biofilm formation is present and an important factor for chronic and asymptomatic avian cholera. Experimental infections of chickens with biofilm deficient P. multocida strain WT X73, proficient biofilm forming P. multocida strain X73ΔhyaD, and proficient biofilm forming clinical isolates 775 and 756 showed that virulence inversely correlated with biofilm formation. Histopathological analysis showed that biofilm forming isolates induced little inflammation in the lungs, heart, and liver, while biofilm deficient isolates induced greater inflammation. Biofilm material was located in pulmonary tissues of chickens diagnosed with chronic avian cholera using FTL staining.. Quantitative real-time PCR for expression of cytokine genes in the spleens of infected chickens indicated that P. multocida induced Th1 and Th17 immune responses during acute and chronic avian cholera. Chickens that succumbed to acute avian cholera after experimental challenge with WT X73 had high levels of INF-ƴ, IL-1β, IL-6, IL-12A, IL-22, IL-17A, and IL-17RA expression in the spleen compared to all other experimental groups. Antibody titers were low, indicating that antibodies may be less important in managing and clearing P. multocida infections.
Ph. D.

Pasteurella multocida serotype A:3 is a gram-negative bacterial pathogen which is one of the causative agents of shipping fever in cattle. In this study, ompH and two fragments of plpE gene (plpEN and plpEC) were cloned from the genomic DNA of P. multocida P-1062 (ATCC 15743, serotype A:3) and plpEN-ompH and plpEC-ompH fusions were constructed. In vitro expression of the genes was shown in HEK-293 cells. Later, full-length plpE gene was cloned and the recombinant proteins were expressed in E. coli and purified. Three DNA vaccine formulations, namely pCMV-ompH, pCMV-plpEN-ompH and pCMV-plpEC-ompH and five recombinant protein based vaccines, PlpEN-OmpH, PlpEC-OmpH, OmpH, PlpEC and PlpE were generated. Recombinant proteins were formulated with at least one of the adjuvants: alum, CpG, alum-CpG, oil based and oil based-CpG. BALB/c mice were immunized with these vaccine formulations and their sera were used for the evaluation of antibody and serum IFN-&gamma
titers. Protective capacities of the vaccines were also evaluated via challenge of mice with 10 LD50 of P. multocida A:3. DNA vaccines induced immune responses, but did not provide protection. All protein vaccine formulations increased antibody levels and CpG containing formulations enhanced serum IFN-&gamma
titers. 100 µ
g of PlpEC-OmpH protein adsorbed on alum adjuvant conferred 40% protection while no protection was obtained with PlpEN-OmpH. Next, the effects of CpG, or its alum and oil based combinations as adjuvants were investigated on PlpEC-OmpH mediated protection. The vaccine formulation composed of PlpEC-OmpH and oil based-CpG adjuvant conferred 100% protection. Finally, the mice were vaccinated with recombinant OmpH, PlpEC and PlpE formulated with oil based-CpG adjuvant. OmpH, PlpEC and PlpE formulations provided 50%, 60% and 100% protection, respectively. These findings implicated that recombinant PlpE and PlpEC-OmpH fusion proteins when formulated with oil based-CpG adjuvant are potent acellular vaccine formulation candidates against shipping fever.

Swine respiratory diseases are responsible for several direct and indirect losses to swine herds. Pasteurella multocida is a frequent bacterium involved in the Porcine Respiratory Disease Complex (PRDC) in finishing pigs. Historically, P. multocida is known as an opportunist agent, causing secondary bacterial pneumonia in pigs after previousMycoplasma hyopneumoniae and/or viral infections. Nowadays, several studies have been shown the possible ability of this bacterium to cause primary infection, leading to death. The aim of this study was to evaluate the anatomopathology and microbiology of pulmonary lesions of samples obtained from animals with clinical respiratory disease andcompare to pulmonary samples obtained at slaughter. Twenty-five lung samples, from 14 herds with clinical respiratory disease, and 19, collected at slaughter, from other 14 herds, in a total of 44 evaluated lung samples were studied. In all lung samples, bacterial isolation was performed, and only samples with pure P. multocida growth were included in the evaluation. P. multocida isolates were tested for antimicrobial sensitivity to 15 drugs, and lung samples were submitted to gross and microscopic evaluation. Also, co-infections with Influenza type A, porcine Circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae were studied using immunohistochemistry (IHC). The antimicrobial sensitivity evaluation showed higher sensitivity rate to ceftiofur and spectinomycin, and some resistance rate to lincomycin and penicillin. P. multocida isolated from lungs collected at slaughter were more sensitive to amoxicillin than isolated from clinical samples (p<0.05). Pleuritis wasmore often observed in lungs of clinical samples than lungs obtained at slaughter (p<0.05). Moreover, there was a numerical trend that indicated that pericarditis, lymphadenomegaly and cavity exudates occurred more often in clinical samples. By IHC, eight samples were positive to Influenza type A, 11 to M. hyopneumoniae, 12 had M. hyopneumoniae and Influenza type A mixed infection, none were positive to PCV2, and 13 were negative to all three of these agents and only tested positive to P. multocida. In these samples, lesions such as pleuritis, pericarditis and lymphadenomegaly were observed only in clinical samples. This finding suggests the ability of these P. multocida isolates to cause primary infection however, it was not possible to determine in this study specific virulence markers.
As doenças respiratórias dos suínos são responsáveis por inúmeras perdas diretas e indiretas para a suinocultura. Dentro do Complexo de Doenças Respiratórias de Suínos (PRDC), Pasteurella multocida é uma bactéria frequentemente envolvida nestes quadros em animais de terminação. Historicamente, a P. multocida é conhecida como um agente oportunista, causador de pneumonias em leitões que apresentaram infecção prévia por outros agentes bacterianos ou virais. Atualmente, cita-se em diversas partes do mundo a capacidade destabactéria de causar infecção primária, e levar os animais à morte. Com isso, o objetivo deste estudo foi fazer uma avaliação anatomopatológica e microbiológica de amostras de pulmão obtidas de animais com doença respiratória clínica e compará-las como amostras obtidas defrigorífico. Avaliou-se 25 amostras, obtidas de 14 rebanhos com quadro clínico respiratório relevante na terminação, e 19 amostras, coletadas ao abate, oriundas de outros 14 rebanhos,totalizando 44 pulmões avaliados. De todas estas amostras fizeram-se cultivos bacterianos, eincluídos no experimento somente os casos com isolamento puro de P. multocida. Estes isolados foram testados quanto à sensibilidade a 15 antimicrobianos, e todas as amostras de pulmão foram examinadas macro e microscopicamente. Ainda, pesquisou-se co-infecções com Influenza tipo A, Circovirus suíno tipo 2 (PCV2) e Mycoplasma hyopneumoniae, através de imuno-histoquímica (IHQ). A avaliação de sensibilidade aos antimicrobianos demonstrou maior taxa de sensibilidade ao ceftiofur e espectinomicina, e maior taxa de resistência à lincomicina e penicilina. Isolados de P. multocida provenientes de pulmões coletados ao abate demonstraram ser mais frequentemente sensíveis a amoxicilina que cepas isoladas de amostras clínicas (p<0,05). Houve maior ocorrência de pleurite em amostrasclínicas (p<0,05), e uma tendência numérica que indicou maior frequência de pericardite, linfadenomegalia e presença de líquidos cavitários também em amostras clínicas. Pela IHQ, oito amostras foram positivas para Influenza tipo A, 11 para M. hyopneumoniae, 12 apresentaram infecção mista de M. hyopneumoniae e Influenza tipo A, e nenhuma foi positiva para PCV2. Treze amostras foram negativas à IHQ para os três agentes testados, e apresentaram somente isolamento de P. multocida. Dentre estas 13 amostras, foi possível observar a ocorrência de pleurite, pericardite e linfadenomegalia, em amostras clínicas, fato que não ocorreu em amostras de frigorífico. Sugere-se, portanto, a sua capacidade de causar quadros respiratórios primariamente. No entanto, não foi possível pesquisar neste estudo,marcadores de virulência que justifiquem este quadro.

Publicación a texto completo no autorizada por el autor
Compara los genomas de las cepas que infectan bovinos y alpacas (36950 y UNMSM, respectivamente) para conocer la genómica estructural de la cepa aislada de alpacas y dilucidar qué proteínas podrían estar relacionadas en la patogénesis en ambos hospederos (bovinos y alpacas). Por ello se secuencia el genoma de P. multocida que infecta alpaca; se realiza el análisis genómico estructural y funcional y posteriormente el análisis comparativo con P. multocida 36950.
Tesis

A pasteurelose é uma das doenças mais comuns do trato respiratório do coelho em criações comerciais e/ou em biotérios de animais destinados à pesquisa biomédica. A bactéria Pasteurella multocida é o patógeno responsável por uma série de manifestações clínicas em coelhos, incluindo rinite crônica, otite média, pneumonia, infecções no trato genital, formação de abscessos pulmonares e cutâneos, conjuntivite e septicemia hemorrágica. Porém, entre 50 e 70 % dos animais podem incubar o organismo de forma assintomática. Os fatores predisponentes para o desencadeamento dos sinais clínicos incluem acúmulo de amônia no ar (má ventilação), prenhez, aparecimento de doenças concomitantes, distúrbios no ambiente de criação ou na manipulação experimental. A doença está presente no Brasil, ocorrendo surtos com relativa freqüência, no entanto, o diagnóstico é feito com base nos sinais clínicos e necropsia. Dessa forma é difícil precisar a extensão dos prejuízos causados pela pasteurelose à cunicultura. Vacinas comerciais específicas contra a pasteurelose em coelhos não estão disponíveis no mercado. A prevenção, ainda que apresente resultados duvidosos, é realizada utilizando-se antibióticos dissolvidos na água, porém este tipo de tratamento normalmente não protege definitivamente os animais. Uma vez que não existem vacinas disponíveis e o tratamento com antibióticos não estabelece proteção contra a pasteurelose, foram desenvolvidos neste trabalho sistemas carreadores das proteínas antigênicas da membrana da P. multocida. Estes sistemas carreadores são formados por lipossomos, já conhecidos pelo seu potencial como imunoadjuvante, e por microesferas lipídicas, responsáveis por apresentar os antígenos às células apresentadoras de antígenos (APC). Inicialmente, foram obtidas colônias puras da bactéria as quais foram cultivadas em meio de crescimento específico (BHI). Os microrganismos foram isolados, rompidos e as proteínas antigênicas foram detectadas por SDS-PAGE e Western Blotting. Estes resultados mostraram que a maioria das bandas protéicas foi reconhecida pelo anticorpo policlonal contra a P. multocida. Visto que tínhamos um pool de proteínas as quais apresentavam antigenicidade, foi realizada uma solubilização incubando frações de membrana da bactéria com SDS 1 %. Este procedimento resultou em um rendimento de solubilização de 85 %. A obtenção dos proteolipossomos foi realizada pelo método da co-solubilização de lipídio, proteína e detergente. Um bom rendimento de incorporação das proteínas em lipossomos parecer estar relacionada com a metodologia utilizada para a remoção do detergente da mistura lipídio:proteína:detergente durante o processo de co-solubilização, e também com a natureza do fosfolipídio utilizado. Os resultados indicaram que a resina Calbiosorb® foi a mais eficiente para a remoção do SDS e, dentre os diversos fosfolipídios testados o que melhor incorporou as proteínas foi o DPPC, com rendimento de incorporação de 93 % e diâmetro médio de 180 nm. Além disso, o SDS-PAGE dos proteolipossomos mostrou que todas as espécies protéicas presentes no extrato bruto solubilizado foram incorporadas nos lipossomos de DPPC. O Western Blotting mostrou que as proteínas incorporadas nos lipossomos continuavam a ser reconhecidas pelo anticorpo policlonal contra a P. multocida. Para os ensaios de imunização foram separados 3 grupos de coelhos: (i) imunizados com lipossomos; (ii) imunizados com extrato bruto solubilizado (EBS); (iii) imunizados com os proteolipossomos. Após 21 dias de imunização com as preparações descritas, os animais foram infectados com 105 ufc de bactéria. Todos os animais vacinados previamente com lipossomos ou EBS foram a óbito enquanto que os animais vacinados com os sistemas de proteolipossomos apresentaram sobrevida de 95 %. Além disso, um grupo controle vacinado com a bactéria atenuada na presença de hidróxido de alumínio como imunoadjuvante apresentou uma sobrevida de apenas 30 %, indicando que a vacina convencional não apresenta uma proteção satisfatória contra a pasteurelose. O soro dos animais vacinados com lipossomo, EBS e proteolipossomos foram coletados semanalmente antes e após a infecção experimental para a detecção da produção de anticorpos IgG, IgM e IgA, utilizando-se a técnica de ELISA. Como esperado, os animais vacinados com lipossomos não apresentaram estimulação de nenhum dos anticorpos específicos para P. multocida analisados. Os animais imunizados com EBS apresentaram um significativo aumento dos níveis de IgG sérico 7 dias após a imunização os quais se mantiveram constantes durante todo o período experimental. Os níveis de IgG no soro de animais imunizados com os proteolipossomos apresentam um aumento 7 dias após a imunização, porém não se mantiveram até o momento da infecção experimental. Após a infecção experimental, os níveis séricos de IgG nos animais imunizados com proteolipossomos apresentam um aumento significativo, enquanto que para os imunizados com EBS houve manutenção dos níveis antes obtidos. A análise de anticorpos IgM específicos para a P. multocida mostram uma produção significativamente maior destes anticorpos para animais imunizados previamente com proteolipossomos que para os animais imunizados com EBS. Além disso, após a infecção experimental, a produção de IgM nos animais imunizados com proteolipossomos continuou sendo estimulada, o que não foi observado para os animais imunizados com EBS. O sistema de proteolipossomos não produz anticorpos IgA sistêmicos específicos para a bactéria, porém após a infecção experimental foi possível observar o aparecimento gradativo deste anticorpo no lavado nasal dos animais, durante as semanas de observação. Os animais previamente imunizados com proteolipossomos sobreviventes da primeira infecção experimental foram observados durante 140 dias e novamente infectados, com nova carga bacteriana. Após a reinfecção a sobrevida destes animais foi de 100 % indicando que o sistema de proteolipossomos foi capaz de gerar uma memória imunológica. A análise conjunta dos resultados obtidos na detecção de anticorpos indica que a proteção proporcionada pelos proteolipossomos contra a pasteurelose é devida a estimulação de anticorpos IgG e, principalmente, de IgM. O outro sistema de delivery de proteínas antigênicas desenvolvido foi o de microesferas lipídicas. Foram experimentados diferentes protocolos, porém o que mais se adequou as nossas condições foi obtido da união e adaptação de duas metodologias descritas na literatura. Estudos de microscopia eletrônica de varredura mostraram que as microesferas lipídicas são formadas quando é utilizado 3 % (p/v) de PVA na formulação. Além disso, marcamos as proteínas com isoticiocianato de fluoresceína e a microscopia revelou a presença de estruturas esféricas fluorescentes, indicando a encapsulação das proteínas na região lipofílica das microesferas. Estudos sistemáticos variando a concentração de óleo, fosfolipídio, proteínas e PVA na formação das microcapsulas permitiram um rendimento de encapsulação de cerca de 99 %. Portanto, no presente trabalho, estabelecemos metodologias de incorporação das proteínas antigênicas em lipossomos constituídos de DPPC e em microesferas lipídicas. Além disso, os sistemas de proteolipossomos apresentaram uma satisfatória propriedade de proteção dos coelhos contra a pasteurelose (frente à infecção experimental com P. multocida) indicando que o sistema aqui proposto pode ser utilizado como vacina, prevenindo a pasteurelose em criações de coelhos comerciais ou destinados à pesquisa biomédica.
Pasteurellosis is a common disease in the respiratory tract of commercial and/or biomedical rearing of research rabbits. The bacterium Pasteurella multocida is the pathogen responsible for a range of clinical syntomes, including chronic rhinitis (snuffles), otitis media, pneumonia, genital infection, pulmonary and cutaneous abscesses, conjunctivitis and hemorrhagic septicemia. However, between 50 and 70 % of the animals can harbour the microorganism asymptomatically. The factors that cause the clinical syntomes include the ammonium accumulation in the air (foul ventilation), pregnancy, another concomitant disease, disorder in the rabbit production environment and experimental manipulation. Outbreaks of this disease occur in Brazil with relative frequency; however diagnosis is generally based on the clinical signals and necropsy. Therefore, it is difficult to estimate the extent of losses caused by pasteurellosis druing cuniculture. However, specific commercial vaccines against pasteurellosis in rabbits are not available and prevention is through the use of antibiotics in drinking water, even though this type of treatment generally does not protect the animals. Initially, pure bacteria colonies were obtained, which were cultivated in specific growing media (BHI). The microorganisms were isolated, lysed and the antigenic proteins were detected by SDS-PAGE and Western Blotting. These results show that most protein bands were recognized by the policlonal antibody against P. multocida. Since this protein pool presented antigenicity, the protein mixture was solubilized by incubating 0,5 mg/ml of the membrane fraction with SDS 1 % (w/v) under constant agitation for 2 hours. This procedure resulted in a 85 % solubilization yield. The proteoliposomes wew formed using a lipid, protein and detergent co-solubilization method. A good yield of protein incorporation in liposomes seems to be related to the methodology used for the removal of the detergent from the lipid:protein:detergent mixture during the co-solubilization process, as well as the nature of the phospholipid used. The results indicated that the Calbiosorb® resin was the most efficient for SDS removal and, among the various phospholipids tested, DPPC best incorporated the proteins, presenting an incorporation yield of 93% and average proteoliposome diameter of 180 nm. In addition, SDS-PAGE of the proteoliposomes has shown that all the proteic species present in the crude solubilized extract were incorporated in the DPPC liposomes. The Western Blotting has shown that the proteins incorporated in the liposomes continue to be recognized by the policlonal antibody against P. multocida. For the immunization assays, three animal groups were separated: (i) rabbits immunized with liposomes; (ii) rabbits immunized with crude solubilized extract (CSE) and (iii) rabbits immunized with the proteoliposomes. After twenty-one days of immunization with the described preparations, the animals were challenged with 105 ufc of bacteria. All animals previously vaccinated with the liposomes or CSE died while the animals vaccinated with the proteoliposomes systems had 95 % survival after the challenge. Moreover, a control group vaccinated with the attenuated bacteria in the presence of aluminum hydroxide as an immunoadjuvant had only 30% survival, indicating that the conventional vaccine does not protect against pasteurellosis. The serum of animals vaccinated with liposome, CSE and proteoliposomes were collected weekly before and after the experimental infection for the detection of IgG, IgM and IgA antibodies production using ELISA. Animals vaccinated with liposomes did not present stimulation of any of the specific antibodies for the P. multocida analyzed. The animals immunized with CSE presented a significant increase in the IgA serum level seven days after the immunization, but these levels were not maintained until the moment of the experimental infection. After the experimental infection, the serum levels of IgG in rabbits immunized with proteoliposomes showed a significant increase, while for those animals immunized with the CSE the levels were maintained. The analysis of IgM antibodies specific for the P. multocida showed a higher production to animals vaccinated with proteoliposomes than for the animals immunized with CSE. Furthermore, after experimental infection, the production of IgM in animals immunized with proteoliposomes continued to be stimulated, which was not observed for those immunized with EBS. The proteoliposome system does not induce IgA systemic antibodies that were specific for the bacterium. However, after the experimental infections it was possible to observe the gradual appearance of IgA in the nasal lavage of the infected animals on the time course of the experiment. Animals previously immunized with the proteoliposomes which survived the first experimental infection were observed during 140 days and re-infected. After the re-infection, the survival of these animals was 100 %, indicating that the proteoliposome system was able to generate a possible immunological memory. The global analysis of the results obtained in the antibody detection indicates that the protection given by the proteoliposome against pasteurellosis is due to the stimulation of antibodies IgG and mainly of IgM. The other delivery system of antigenic proteins developed during this work is of lipidic microspheres. Different protocols were tried, but the one which was more adequate to our experimental conditions was elaborated from joining and adapting two methodologies described in literature. Scanning electron microscopy studies have shown that the lipidic microspheres are formed when 3 % (w/v) of PVA is used in the formulation. Furthermore, we have marked the proteins with fluorescein isothiocyanate and the microscopy revealed the presence of fluorescent spherical structures which indicated the encapsulation of the proteins in the lipophilic region of the microspheres. Systematic studies varying the concentration of oil, phospholipid, proteins and PVA in the microcapsules formulation has given a yield of encapsulation of 99%. We have established methodologies of incorporation of the antigenic proteins in liposomes constituted of DPPC and lipidic microspheres. Moreover, the proteolipossome systems have shown a satisfying property of protection of rabbits against pasteurellosis in face of the experimental challenge with P. multocida indicating that the system proposed here can be used as a vaccine to prevent the pasteurellosis either in commercial or biomedical research rearing of rabbit.