Thesis (M.S.)--University of Wisconsin--Madison. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 43-47).

Thesis (M.S.)--University of Wisconsin--Madison, 1968. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

[No abstract submitted] / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Brucella abortus is the etiological agent of bovine brucellosis, a zoonotic disease that can be transmitted to humans through direct contact with infected animals or consumption of contaminated food products. Current serological tests used to identify infected animals rely on the detection of antibodies to O-antigens of the smooth lipopolysaccharide (sLPS) of B. abortus. Due to the presence of structurally similar O-antigens of bacteria such as E. coli O:157 and Yersinia enterocolitica O:9, these tests can produce false-positive results, which requiring alternative protein target antigens. We hypothesize that through comparative genomics and bioinformatics analysis of all ORFs within the B. abortus genome, followed by profiling of the humoral immune responses to surface and extracellular proteins, novel protein antigens with diagnostic potential may be discovered. In this study, the genomes of thirteen strains were analyzed using a subcellular localization prediction database (PSORTb) to identify proteins in the outer membrane and extracellular space. A total of 100 ORFs coding for such proteins including known immunogenic proteins reported in literature were identified and selected for recombinant protein expression using high-throughput in vivo cloning and in vitro transcription/translation strategies. The in vitro expression of 67 of these candidates has been successfully demonstrated. These recombinant B. abortus proteins were subsequently probed with E. coli pre-adsorbed sera from infected animals for the identification of immunoreactive protein antigens. Ten unique candidates were demonstrated to be antigenic and have the potential for diagnostic applications. This study illustrates a unique, high throughput strategy to express and screen proteins of a bacterial pathogen for novel diagnostic antigen discovery.

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Tuberculosis, caused by Mycobacterium tuberculosis, is one of the most prevalent infectious diseases inflicting humankind. The World Health Organization estimates that one third of the world's population, approximately 2.2 billion people, is infected with TB with a mortality of 1.7 million people annually. Currently, the WHO estimates that each year more than 9 million people develop TB. Bacille Calmette-Guérin (BCG), an attenuated strain of M. bovis, is the only licensed TB vaccine in the world. Clinical studies have shown childhood vaccination with BCG to be protective against disseminating and meningeal forms of TB. However, the efficacy of BCG against pulmonary TB in adults has been variable and inconsistent (0-80%). The objective of this study is to develop and test the efficacy of the B. abortus vaccine strain RB51 as a platform for expression of M. tuberculosis antigens (Ag85B, ESAT6 and Rv2660c) and induction of a protective immune response against M. tuberculosis and B. abortus challenge in mice. Here we report the construction of two recombinant strains of B. abortus vaccine strain RB51 capable of expressing mycobacterial antigens Ag85B, ESAT6 and Rv2660c. Our studies show that expression of mycobacterial antigens in strain RB51 lead to induction of antigen-specific immune responses characterized by secretion of IgG2a antibodies as well as of IFN- and TNF-α. Mice immunized with a combination of two strains of RB51 in equal numbers, one carrying Rv2660c-ESAT6 and another carrying Ag85B, led to a 0.90 log reduction in CFU burden with significance nearly reaching borderline (p = 0.052). However, when mice were primed with the same strains of RB51 and boosted with proteins Ag85B and ESAT6, a significant level of protection (1 log reduction) compared to the PBS vaccinated group was achieved. The protection levels conferred by this vaccination strategy was similar to that conferred by BCG vaccine. In conclusion, we have shown that recombinant RB51 strains expressing mycobacterial protective antigens result in stimulation of antigen specific immune response without altering the vaccine efficacy in protecting against the more virulent strain of B. abortus 2308. These recombinant vaccines could potentially be used to protect against M. tuberculosis infection. / Ph. D.

Through chemical analysis and ion exchange chromatography of watersoluble antigens, this investigation supports the view that the majority of differences between the biotypes are quantitative. It was also found that strains demonstrate distinct, qualitative differences when compared to the attenuated strain 19 by immunodiffusion and thin-layer polyacrylamide gel, isoelectric focusing. These differences include the presence of antigens on virulent strains that are absent on strain 19. In addition, one antigen absent on strain 19, was found common to each virulent biotype. Finally, the results from immunodiffusion experiments, employing adsorbed and non-adsorbed immune globulins, indicate that at least some water-soluble antigens are exposed on the cell surface and that their distribution among the biotypes varies.

Determina la incidencia y aislamiento de especies de Brucella en pacientes clínicamente sintomáticos y mediante análisis de aglutinaciones, hemográmas y su respectivo cultivo de muestras de sangre. Los casos investigados fueron seleccionados entre los pacientes concurrentes al Servicio Académico Asistencial de Análisis Clínicos de la Facultad de Farmacia y Bioquímica de la Universidad Nacional Mayor de San Marcos, entre los meses de enero a agosto de 1992. Analiza 172 muestras de pacientes. Obtiene positividad inmunológica en 26 pacientes (15:11%) y positividad bacteriológica mediante hemocultivos en cuatro pacientes. Con mayor prevalencia en el grupo etario de 20 a 30 años. Para el aislamiento de la Brucella, se ensayaron hemocultivos en medios bifásicos, elaborados en base a infusión de papa, tanto para el medio sólido como para el medio líquido, lográndose aislar dos serotipos de Brucella: tres cepas de Brucella melitensis y una cepa de Brucella abortus. Las cepas de Brucella, representan el 2.33% del total de pacientes estudiados, de los cuales dos son del sexo femenino y dos son del sexo masculino. / Trabajo de suficiencia profesional

Les Brucella sont des bactéries intracellulaires responsables de la brucellose, une zoonose mondiale qui cause des avortements chez les animaux, entrainant d'énormes pertes économiques dans les élevages, et des problèmes de santé de longue durée chez l'Homme. Contrairement aux animaux, il existait jusqu’à présent peu de preuves que les infections à Brucella pouvaient causer des complications obstétriques chez la femme. Des récentes études épidémiologiques ont cependant démontré une augmentation significative des risques de complications (fausses couches, mort in utéro, accouchement prématuré) chez les femmes enceintes infectées par Brucella. De plus, il a été montré que plusieurs espèces zoonotiques de Brucella sont capables d’infecter des cytotrophoblastes (CTB) et des trophoblastes extravilleux (EVT) humains, deux types de cellules ayant des fonctions immunitaires et hormonales essentielles pendant le développement du placenta. Dans ce travail, nous avons étudié les conséquences de l’infection des trophoblastes humains par Brucella, du côté du pathogène mais aussi du côté de l’hôte. Nous avons évalué le comportement intracellulaire de différentes souches de Brucella, représentant différentes espèces, hôtes ou symptômes associés. Nous n'avons trouvé aucune corrélation entre la capacité de réplication dans les trophoblastes et l’association des souches avec des complications obstétricales chez leur hôte respectif. Nous nous sommes également intéressé à des souches récemment isolées chez des babouins, après une infection placentaire ayant causé la mort in utéro de leur fœtus. Nous avons montré que ces souches sont capables d’infecter les trophoblastes humains et affectent certaines de leurs propriétés qui sont essentielles lors du développement placentaire. Nous avons également commencé à caractériser des structures intracellulaires atypiques dans lesquelles Brucella semble pouvoir survivre dans certains trophoblastes. Du côté hôte, nous avons analysé le rôle de la protéine eucaryote CD98hc dans l'infection les trophoblastes. Nous avons montré que CD98hc est importante pour l'infection des trophoblastes humains par Brucella, comme cela avait été montré précédemment dans d'autres types cellulaires, et que l’infection modifie le niveau de cette protéine dans les trophoblastes. L'infection des trophoblastes humains par Brucella pourrait donc altérer leurs fonctions au cours du développement placentaire, entraînant ainsi des complications pendant la grossesse.Les résultats obtenus dans ce travail contribuent à une meilleure compréhension des mécanismes pouvant causer des complications obstétricales chez la femme enceinte infectée par brucella et fournissent des informations importantes pour la prise en charge clinique de la brucellose pendant la grossesse. / Brucella are intracellular bacteria responsible for brucellosis, a worldwide zoonotic disease associated with infectious abortions in animals, which causes huge economical losses in the livestock industry and long lasting health problems in humans. In contrast to animals, evidence of Brucella infections cause obstetric complications in humans is scarce. However, epidemiological studies have shown significant increases in the risk of adverse pregnancy outcomes (miscarriage, stillbirth, preterm delivery) in pregnant women infected with Brucella. Moreover, several zoonotic Brucella species were shown to infect efficiently human cytotrophoblasts (CTB) and extravillous trophoblasts (EVT), two types of cells with essential immune and hormonal functions during placental development. In the present study, we studied the effect of Brucella infection in human trophoblasts, from both the bacterial and the host sides. We evaluated the intracellular behavior of different Brucella strains, representing different species, hosts or associated symptoms. We found no correlation between the bacterial replication rate in trophoblasts and whether the strains were associated with pregnancy complications in their respective host. Importantly, we show that strains isolated from female baboons after stillbirth can infect human trophoblasts and affect some of their properties that are essential during placental development. We also started the characterization of atypical intracellular structures in which Brucella seem to be able to survive in certain types of trophoblasts. From the host side, we analyzed the role of the eukaryotic protein CD98hc in trophoblast infection. We found that CD98hc is important for infection of trophoblasts by Brucella, as shown previously in other cell types, and that infection affects the level of the protein in trophoblasts. Infection of human trophoblasts by Brucella could thus affect their function during placental development, leading to complications in pregnancy.The results obtained in this work contribute to a better understanding of the mechanisms that could lead to obstetric complications in Brucella infected pregnant women and provide important information for the clinical management of brucellosis during pregnancy.