Although it is known that cell-mediated immunity (CMI) plays a key role in protection against brucellosis, the exact immune mechanisms leading to protection are still not fully understood. Better understanding of the mechanisms would help in the development of a human Brucella vaccine and help in improving animal vaccines. In this research, B. abortus strain RB51 and a closely-related, nonpathogenic Ochrobactrum anthropi (strain 49237) bacterium were used to study the immune response against brucellosis in mice. Both O. anthropi strain 49237 and recombinant strain 49237 expressing Brucella protective antigen copper-zinc superoxide dismutase (Cu/Zn SOD) induced a mix of Th1 and Th2 type immune responses but failed to provide protection against virulent Brucella challenge. After changing the immune response to a predominantly Th1 type of response using CpG oligonucleotides as an adjuvant, both strains provided protection with the recombinant strain inducing significantly higher protection. It was also demonstrated that vaccination with strain RB51 induced Th1 immune responses characterized by high interferon-gamma (IFN-g) production with no interleukin-4 (IL-4) secretion as well as high IgG2a and minimal IgG1 production. A colorimetric cytotoxic T lymphocytes (CTL) assay was developed to demonstrate that strain RB51 induced an antigen-specific CTL reaction that probably plays an important role in protection. The results suggest that optimal protection against brucellosis requires IFN-g-secreting T cells and antigen-specific CTLs. Recombinant strain RB51 overexpressing Brucella Cu/Zn SOD and simultaneously expressing mycobacterial 85A antigen induced higher IFN-g production and CTL activity than the parent RB51 strain. The combined results suggest that the recombinant O. anthropi strain could be used as a human vaccine against brucellosis and that the recombinant RB51 strain could be used as an effective vaccine against both brucellosis and tuberculosis in animals. / Ph. D.

Made available in DSpace on 2016-08-29T15:38:41Z (GMT). No. of bitstreams: 1 tese_7206_Outbreak of Ochrobactrum anthropi endophthalmites following cataract surgery.pdf: 114449 bytes, checksum: c89d6abc157b93a9d71ed933dca2014c (MD5) Previous issue date: 2013-11-11 / Endoftalmite infecciosa após cirurgia muitas vezes progride para deficiência visual significativa e irreversível. A tese descreve um surto de endoftalmite por Ochrobactrum anthropi ocorrido após cirurgia de catarata e propõe um novo protocolo de esterilização para minimizar o risco de novos casos. Prontuários de pacientes com diagnóstiico firmado de O. anthropi por cultura ou com achados clínicos sugestivos durante o surto foram revisados. Sete casos de endoftalmite por O. anthropi foram confirmados entre 24 de Julho e 10 de novembro de 2010. A causa mais provável do surto foi a contaminação da tubulação da máquina de facoemulsificação. Após a introdução do novo protocolo de esterilização, não houve mais casos de endoftalmite, independente da causa, em mais de 1000 procedimentos subsequentes.

The species of Brucella were isolated and characterized almost 120 years ago and their genomes sequenced for almost 4 years. Compared to other bacterial pathogens relatively, little is known about the factors contributing to their persistence in hosts and multiplication within phagocytic cells. Also, many aspects of the interactions between Brucella and its host remain unclear. Molecular characterization of intracellular survival processes of Brucella will provide guidance for additional prevention and control measures. One of the features that distinguishes Brucella is that they do not express classic virulence factors. Thus identification of virulence factors has been elusive and some of the identified virulence genes are putative. Disruption of putative virulence genes and studying the consequent effect on attenuation in cell lines or mouse models is a widely used method. However, in most cases it is not apparent whether the mutated genes encode virulence factors or merely affect normal metabolic or biological functions. Some mutations in Brucella can be compensated by redundancy or backup mechanisms. One method for identifying putative virulence genes involved in pathogenesis is to express these genes in a nonpathogenic host and isolate recombinants with increased virulence or survival ability either in cell culture or animal model. We hypothesize that over-expression of Brucella putative virulence genes in the non-pathogenic and close phylogenic relative Ochrobactrum anthropi should enhance its survival in infection models in vivo. O. anthropi is one of the closest Brucella relatives based on DNA, rRNA, and protein analyses but it is unable to establish chronic infection and considered as opportunistic pathogen that, under certain circumstances, may produce disease in immunocompromised humans. Therefore, we established enhanced expression system in Brucella and Ochrobactrum to identify B. suis virulence genes. We created an enhanced expression system that can be used for cloning and expression of heterologous genes in Brucella and Ochrobactrum. We studied the transcriptional activity of several promoters and created some tools to enhance the expression, detection and purification of Brucella recombinant protein in Ochrobactrum. The presumable importance of alkyl hydroperoxide reductases encoded by ahpC and ahpD genes and their contribution to intracellular survival of Brucella were studied by over-expressing them. The recombinant O. anthropi expressing B. suis ahpC and ahpD genes were able to resist in vitro killing by H2O2 and or cumene hydroperoxide and survived longer in the macrophage J774 A.1 cell line. The control O. anthropi was cleared from BALB/c mice in five days while the recombinants were recovered from spleens, livers and lungs of infected mice up to eight days post-infection. We tested the contribution of B. suis cyclic glucan synthetase gene (cgs) to virulence by over-expressing it in O. anthropi. We studied the ability of the recombinant O. anthropi to resist killing in vitro and in vivo. We generated evidence that B. suis cgs when over-expressed in O. anthropi increased the amount of cyclic glucans synthesized and accumulated in the periplasmic space. This accumulation changed the virulence of the microorganism from a soil bacterium that cleared from mice in less than five days into a pathogenic organism that could survive up to 9 days and at higher doses killed the mice. In summary, several vectors have been constructed for gene expression and protein purification in Brucella and Ochrobactrum. Novel useful tools for enhancement of heterologous gene expression were created and demonstrated to work in Brucella and Ochrobactrum. Brucella putative virulence genes were studied in Ochrobactrum using the newly constructed vectors and tools. Ochrobactrum as a gain of function model for studying putative virulence genes of intracellular pathogens in general and for Brucella in particular proved to be a very useful model. / Ph. D.