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.

Brucella canis causes canine brucellosis in dogs inducing mainly contagious abortion. Diagnosis of B. canis is based on bacterial isolation that is timeconsuming and inconsistent; serological tests (more than one test) that is ambiguous and lacks specificity; and PCR that may lack sensitivity as bacteraemia may not be constant. Since bacteraemia of B. canis develops 7-30 days after infection, often resulting in a sustained bacteraemia, PCR was investigated for the detection of B. canis in whole blood of dogs. The PCR sensitivity was validated to detect 3.8 fg Brucella DNA mixed with dog DNA as well as 1 x 102 cfu/ml B. canis in dog blood (mock infection) using primers (ITS66 and ITS279) that amplifies the 16S-23S ribosomal DNA intergenic spacer (ITS) region. The PCR assay for the detection of B. canis in whole blood samples was compared with bacterial isolation, serological tests, which include the rapid slide agglutination test (RSAT), 2-mercaptoethanol RSAT (2ME-RSAT) and imunochromatographic assay (ICA). These techniques were used to test 56 dog samples obtained from the Michangulene and Mafavuca villages at the municipality of Changalane, in District of Namaacha in Maputo, Mozambique for B. canis. No B. canis was isolated from dog blood using the classical microbiology isolation and PCR. A sample was only presumed positive if both the 2ME-RSAT and ICA tested positive. None of the samples in this study tested positive using this criterion for serological testing. Results of this study indicated that B. canis was not present in the 56 dogs sampled in the Maputo region of Mozambique using bacteriology, PCR and serological tests (RSAT, 2ME-RSAT and ICA). Due to the discrepancy between serological tests we cannot conclude that B. canis is not present in the Maputo region of Mozambique. In future the accuracy of the serological tests, bacteriology and PCR assay should be assessed using experimentally infected B. canis dogs over a period followed by a surveillance study in Mozambique that includes urine, semen and blood samples collected from dogs. / Dissertation (MSc)--University of Pretoria, 2010. / Veterinary Tropical Diseases / Unrestricted

Brucella abortus is a facultative, intracellular, zoonotic pathogen that resides inside macrophages during infection. This is a specialized niche where B. abortus encounters various stresses, such as acidic conditions and reactive oxygen species, as it navigates through the macrophage. In order to survive this harsh environment, B. abortus utilizes post-transcriptional regulation through the use of small regulatory RNAs (sRNAs). sRNAs bind to messenger RNA (mRNA) targets via complementary base pairing. sRNAs are a class of regulatory molecules in bacteria that elicit rapid post-transcriptional regulation. sRNA-mRNA binding can positively or negatively influence gene expression. Positive regulation can occur through sRNA binding to protect the mRNA from RNases. sRNA binding can also alleviate the secondary structure and reveal the ribosomal binding site. Alternatively, sRNA-mRNA interactions can have negative consequences on gene expression through degradation via RNases or sRNA binding can occlude the ribosomal binding site. Although some sRNAs have been discovered in B. abortus, few have been characterized in regards to virulence. In this study, B. abortus was stressed in conditions relevant to the macrophage, including, including low pH, oxidative stress, and nutrient limitation. Transcriptomic analysis revealed high levels of transcripts in intergenic regions, a hallmark of sRNAs, which led to the discovery of VcrS for virulence and cell wall regulating sRNA. A ΔvcrS was engineered and this mutant was used to infect both naïve murine macrophages, as well as BALB/c mice. Both virulence studies demonstrated significantly decreased bacterial recovery of ΔvcrS compared to the wildtype strain. Quantitative proteomics revealed that one protein, BAB1_1454, is 30-fold over-produced in ΔvcrS compared to wildtype. This essential protein encodes MurF, which catalyzes the final cytoplasmic step of generating the mura-pentapeptide precursor for peptidoglycan synthesis. VcrS is hypothesized to interact with murF mRNA and interfere with translation initiation. Sequence data indicates a putative 6 nucleotide motif in VcrS that has complementarity to the ribosomal binding site of murF. Identification of the binding site and further characterization of VcrS will showcase the importance of sRNA regulation in the virulence of B. abortus. / Master of Science / Brucella abortus is a bacterial pathogen that primarily infects cattle but is also transmitted to humans. Human disease most commonly results from the consumption of unpasteurized milk and milk products. Human brucellosis has very limited treatment options, with a high incidence of disease relapse. B. abortus survives and replicates within immune cells, which create a harsh environment. However, the bacteria are able to sense and adapt to survive and replicate within these immune cells, maintaining a chronic infection. A better understanding of the adaptation process B. abortus utilizes to survive within the human host can lead to improvement of treatment options. The present work characterizes a novel regulatory small RNA- VcrS, which was found required for survival and replication inside immune cells

A soluble acid-heat extracted Brucella vaccine: Immunological and physiological studies in guinea pigs, rabbits and calves. 143 p. Dissertation. 1959. -- A soluble-type vaccine was prepared by the acid-heat extraction of Brucella abortus Strain 2 308 and its metabolic by-products in Stuart• s medium. A comparison was made, in guinea pigs, of 2-day-old and 13-day-old cultures for the preparation of the immunogenic agent. Further comparisons were made in guinea pigs and calves of the 13-day-old culture and cell-free 13-day-old culture vaccine. The agent made from the 2-day-old culture produced no significant protection against various challenge levels of virulent Br. abortus Strain 2308. The agent prepared from 13-day-old cultures not only produced significant protection against homologous strain challenge but, produced insignificant serum-agglutination titers at effective dosage levels. The 13-day-old whole and cell-free culture vaccines gave similar results in guinea pigs. In calves the 13-day-old whole-culture vaccine produced higher transient serum-agglutination titers than the 13-day-old cell-free culture vaccine. Protective studies in calves were inconclusive due to inadequate infection in the control animals. The vaccine was shown to be essentially protein in nature and contained two distinct fractions on paper electrophoretic examination. The less mobile fraction apparently contained the agglutinogenic material and the more mobile fraction apparently contained the immunogenic material. Removal of the Brucella cells, prior to acid-heat extraction, decreased the less mobile fraction by more than one-half. The degree of serum agglutinin titer response was apparently contingent on this fraction, which indicated the agglutinogens were an index of metabolic byproducts, but this did not imply that they were an index of protection. Paper electrophoretic serum-protein patterns of 15 male and 15 female rabbits were studied. It was determined that: the serum fraction percentages of normal rabbits showed little variation, with no differences between sex or breed. The various pathologic conditions were indicated first by serum-protein patterns, and later diagnosed by histopathological examination of necropsy material. An additional 14 rabbits with a natural Eimeria stiedae infection were also investigated. The use of paper electrophoresis as an aid in the selection of normal animals for experimental investigation was demonstrated. The 13-day-old whole culture vaccine was employed in rabbits to study serum-agglutinin titer response. The results indicated that the maximum gamma globulin response followed the peak agglutinin response by seven to twenty-one days, this indicated a secondary response the nature of which was not determined. Female rabbits responded to the vaccine with higher initial titers than males and the titer decline was one and one-half times more rapid in the males than. in the females. Sex was shown to be the most significant factor in this finding. Castration and ovario-hysterectomy indicated that the above results could be reversed when an estrogenic hormone was given to the castrates and when testosterone was given to the ovario-hysterectomized animals. Insufficient data was available to elucidate the role of steroid hormones in the serum-agglutinin titer response. / Ph. D.

Comparative analysis of multiple genomes presents many challenges ranging from management of information about thousands of local similarities to definition of features by combination of evidence from multiple analyses and experiments. This research represents the development stage of a database-backed pipeline for comparative analysis of multiple genomes. The genomes of three recently sequenced species of Brucella were compared and a superset of known and hypothetical coding sequences was identified to be used in design of a discriminatory genomic cDNA array for comparative functional genomics experiments. Comparisons were made of coding regions from the public, annotated sequence of B. melitensis (GenBank) to the annotated sequence of B. suis (TIGR) and to the newly-sequenced B. abortus (personal communication, S. Halling, National Animal Disease Center, USDA). A systematic approach to analysis of multiple genome sequences is described including a data model for storage of defined features is presented along with necessary descriptive information such as input parameters and scores from the methods used to define features. A collection of adjacency relationships between features is also stored, creating a unified database that can be mined for patterns of features which repeat among or within genomes. The biological utility of the data model was demonstrated by a detailed analysis of the multiple genome comparison used to create the sample data set. This examination of genetic differences between three Brucella species with different virulence patterns and host preferences enabled investigation of the genomic basis of virulence. In the B. suis genome, seventy-one differentiating genes were found, including a contiguous 17.6 kb region unique to the species. Although only one unique species-specific gene was identified in the B. melitensis genome and none in the B. abortus genome, seventy-nine differentiating genes were found to be present in only two of the three Brucella species. These differentiating features may be significant in explaining differences in virulence or host specificity. RT-PCR analysis was performed to determine whether these genes are transcribed in vitro. Detailed comparisons were performed on a putative B. suis pathogenicity island (PAI). An overview of these genomic differences and discussion of their significance in the context of host preference and virulence is presented. / Master of Science

The effect of dietary copper (Cu) supplementation with Cu-Sulfate (CuSO₄) or Cu-Lysine (CuLy) on Cu status and bovine monocyte function was evaluated through a series of experiments. Initially, two in vitro techniques, immunomagnetic (IM) and culture flask adherence (CF), were compared for isolation of a viable, homogeneous monocyte population. The CF technique for monocyte isolation resulted in both a greater number of cells exhibiting phagocytic activity, as well as, an increased phagocytic capacity compared to monocytes recovered by the IM technique. Culture flask adherence appears to be an efficient technique for isolation of a viable, homogeneous population of bovine monocytes. Copper status and monocyte function were evaluated in beef calves supplemented with Cu over a 2 year study period. Fifty-four weaned calves were allotted to one of three Cu treatment groups in a 150 d feeding trial, Plasma Cu concentration was increased in CuLy-supplemented calves over controls and CuSO₄-supplemented calves on d 42, 84, and 126. Calves supplemented with Cu had increased ceruloplasmin activity on d 84, 126, and 150 as compared to controls. Hepatic Cu measured on d 150 was decreased in controls compared to Cu-supplemented calves. Monocyte cell number and function from CuLy-supplemented calves showed increased phagocytosis on d 84 and 126 and increased oxidative burst on d 42 and 126 compared to controls. Dietary Cu supplementation was repeated using 45 calves in a 120 d study. CuLy-supplemented heifers had increased major histocompatibility complex (MHC) class II expression on d 68, 82 and 110 compared to CuSO₄-supplemented and control group heifers. Heifers supplemented with Cu had increased plasma Cu concentrations on d 82 and 110 compared to controls. The effect of vaccination on monocyte function was evaluated in Cu-supplemented beef heifers. Vaccination with B. abortus Strain 19 increased monocyte oxidative burst, phagocytic activity, and MHC class II expression in heifers. Copper supplementation and source of Cu supplement influenced monocyte response to vaccination. Monocyte response appeared to be higher in CuLy-supplemented heifers after vaccination compared to CuSO₄-supplemented and control heifers. / Ph. D.

Rhizobiales encompass a diverse group of microbes, ranging from free-living, soil-dwelling bacteria to disease-causing, intracellular pathogens. Although the lifestyle of these organisms vary, many genetic systems are well conserved. One system, named the AbcR/VtlR system, is found throughout rhizobiales, and even extends to bacteria in other orders within the Alphaproteobacteria. The AbcR sRNAs are an example of sibling sRNAs, where two copies of the abcR gene are typically present in the genome. The AbcRs are involved in the negative regulation of ABC-type transport systems, which are important components for nutrient acquisition. Although the AbcRs share several features amongst organisms, major differences can be found in their functional and regulatory redundancy, the targets they regulate and how they regulate them. Specifically, one major difference in the AbcRs lies in the nucleotide sequences utilized by the sRNAs to bind mRNA targets. In the present studies, the regulatory mechanisms of the AbcR sRNAs were further characterized in the mammalian pathogen Brucella abortus, and the full regulatory profiles of the AbcRs were defined in the plant pathogen Agrobacterium tumefaciens. As mentioned above, the AbcR sRNAs are important for the proper regulation of nutrient-acquiring transport systems in the Rhizobiales. Since these sRNAs are critical to the lifestyle of a bacterium, proper regulation of this system is key to survival. A LysR-type transcriptional regulator, named VtlR, was found to be the bonefide transcriptional activator of abcR1 in B. abortus. Furthermore, VtlR has been shown to be a key component in host interactions in several rhizobiales. The preset work has shed light on the evolutionary divergence of this regulator in bacteria, and further defined the regulatory capacity of VtlR in Agrobacterium. Overall, the studies described here have made significant advances in our knowledge of the AbcR/VtlR-regulatory systems in the Rhizobiales, and have further defined this system as being a vital part of host-microbe interactions. / PHD / Understanding the genetic systems utilized by microbes to cause infection is key for developing therapeutics that can be administered to fight against them. Moreover, identifying and characterizing these essential microbial systems can be exploited for the development of drugs to target and shut down these systems, thus causing cell death. The present work took a basic molecular biology approach and characterized a highly conserved genetic system, named the AbcR/VtlR system, in two pathogenic bacteria: the plant pathogen Agrobacterium and the mammalian pathogen Brucella. Overall, the work described here shows this system to be an important component in acquiring nutrients for the microbe, and, most importantly, found the AbcR/VtlR system to be essential for host-microbial interactions.

Memoria para optar al Título Profesional de Médico Veterinario / En el presente trabajo se desarrolló y validó un ensayo inmunoabsorbente ligado a un enzima (ELISA), usando antígeno lipopolisacárido rugoso (LPS-R) de la cepa RB51 de Brucella abortus para el diagnóstico de infección con B. ovis en ovinos. Se probaron 2 tipos de placas de poliestireno NUNC 69620 y Maxisorp, 2 tipos de tampones de distinto pH con 2 diferentes conjugados: policlonal anti IgG-ovina (Sigma A3415) y monoclonal anti IgG-caprino/ovino (Sigma A9452), las diluciones del antígeno y del conjugado del trabajo se obtuvieron mediante una titulación en tablero de ajedrez. El ensayo se validó utilizando la placa NUNC 69620 con una sensibilización del antígeno 1:50 en el tampón carbonato-bicarbonato y el uso del conjugado monoclonal, en 55 sueros de ovinos positivos a examen clínico y a la prueba de reacción en cadena de la polimerasa (PCR) y 338 sueros de ovinos provenientes de un rebaño libre de la enfermedad, confirmados por medio de fijación del complemento (FC). Para comparar las placas, las densidades ópticas (DO) fueron llevadas a porcentajes de positividad (PP), el punto de corte fue un 39,5% utilizando la metodología “Receiver-Operator Characteristic” (ROC), estimando la sensibilidad y la especificidad en un 92.7% y un 98,5% respectivamente. Estos resultados permiten establecer que el ELISA desarrollado sea una alternativa real, para el diagnóstico serólogico de B. ovis en el país

La capacité de Brucella suis, un microorganisme strictement aérobique, a s'adapter aux taux d'oxygène faible est un processus essentiel pour la virulence et la persistance bactérienne. Le manque d'oxygène est une condition hostile à laquelle les bactéries sont confrontées lors de la pénétration de l'hôte et pour établir leur niche replicative et la phase de persistance. Cette bactérie possède plusieurs mécanismes par laquelle elle s'adapte à cette condition. Elle peut utiliser un régulateur transcriptionel de la famille de FnrN dépendant de l'oxygène, deux cytochromes oxydases de haute affinité pour l'oxygène et une voie complète de denitrification pour résister au manque d'oxygène. Ce travail a démontré que la respiration oxydative et la denitrification peuvent être simultanément utilisés par B. suis sous microaerobiosis. RegBA, un système à deux composants chez B. suis, a été aussi identifié nécessaire dans l'adaptation bactérienne au manque d'oxygène. Ce dernier a été démontré à coordonner le contrôle des systèmes respiratoires précédemment évoqués. Un schéma de régulation global chez B. suis des voies respiratoires par le régulateur transcriptionel RegA a été suggéré : lors de la variation de l'état redox, la cytochrome bd oxidase jouerait un rôle dans la transmission d'un signal à RegB le senseur de la histidine kinase. De plus, RegA a été identifié essentiel pour la persistance de B. suis in vivo chez la souris dans les organes avec des teneurs faible en oxygène. RegA est supposé être impliqué dans l'installation de la phase de persistance bactérienne durant l'infection chronique. Cette étude a aussi identifié le rôle potentiel de RegA dans la régulation de nombreux gènes impliqués durant la phase de persistance. En utilisant une analyse transcriptomique, comparant les taux d'hybridation chez les souches sauvage et muté dans un modèle in vitro qui imite les conditions d'une infection chronique correspondant a un manque de nutriment et d'oxygène, 447 gènes avec un taux d'hybridation ≥ 2, ont été détectés réguler par RegA. Chez la souche sauvage, 45% et 55 % des gènes étaient régulés et réprimés par RegA chez la souche sauvage, respectivement. 14% des résultats du transcriptome a été choisi pour la validation génétique par RT-qPCR. RegA induit l'expression de gènes impliqués dans le métabolisme d'énergie y compris des gènes de la respiration oxidative, ce qui confirme qu'il interagit dans l'adaptation bactérienne au manque d'oxygène. RegA réprime des gènes impliqués dans la réplication d'ADN, la biogenèse de l'enveloppe et la division cellulaire, de même certains gènes dans le métabolisme d'énergie, ce qui suggère son effet sur la multiplication et l'adaptation bactérienne à l'hypoxie qui existe durant la phase de persistance. RegA a été démontré a réprimer les facteurs de virulence l'operon virB ainsi que son régulateur VjbR. De plus, cette étude a évalué le rôle de deux gènes BR1614 et BR1510 régulés par RegA et impliqués dans le métabolisme des acides gras. Dans les expériences in vivo chez la souris ont démontré que les deux gènes sont essentiels pour la survie, la multiplication et la persistance bactérienne. En conclusion, RegA régule, directement et indirectement, l'expression de gènes qui codent pour la traduction, la transcription, la production d'énergie et la conversion, la réparation d'ADN et de protéine. Ces résultats suggèrent un rôle majeur pour RegA dans la persistance bactérienne pendant la brucellose. 12% du génome de B. suis est sous le contrôle de RegA ce qui indique qu'il est un régulateur global comme son PrrA d'homologue dans Rhodobacter sphearoides. / The capacity of Brucella suis, a strictly aerobic microorganisms, to adapt to low oxygen level is of high importance as it is a required and an essential process for bacterial establishment of virulence and persistence. Oxygen deficiency is a hostile condition to which bacteria are faced when they penetrate the host and reach their replicative niche as well as the persistence phase. This bacterium possesses several mechanisms that answer remarkably to this condition. It can use an oxygen-dependent transcriptional regulator of the FnrN family, two high-oxygen-affinity terminal oxidases, and a complete denitrification pathway to resist various conditions of oxygen deficiency. This work has demonstrated that the oxidative respiration and denitrification can be simultaneously used by B. suis under microaerobiosis. RegBA, a two component systems in B. suis, was also identified to be necessary in bacterial adaptation to oxygen deficiency as it was demonstrated to coordinate the control of the respiratory systems mentioned previously. A scheme for global regulation of B. suis respiratory pathways by the transcriptional regulator RegA was suggested: under redox variation, the cytochrome bd ubiquinol oxidase would play a role in the transmission of a signal to the histidine sensor kinase RegB. RegA in addition was found to be essential for B. suis persistence in vivo in mice within low oxygenated organs. RegA is thus assumed to be involved in the establishment of bacterial persistence during chronic infections. This study also investigated the potential control of RegA in the regulation of numerous genes during the persistence phase. By using a microarray assay comparing wild-type and ∆regA mutant strains, in an in vitro model that mimic the conditions of a chronic infection corresponding to nutrient and oxygen deficiency, 447 genes with a cutoff of the level of hybridization intensities ≥2, were detected regulated by RegA. In the wild-type strain, 45% and 55 % of the genes were up-regulated and down-regulated in wildtype strain, respectively. 14% of the microarray results were selected for genetic validation by RT-qPCR. RegA induced the expression of some genes involved in energy metabolism including the oxidative respiratory genes confirming that it interacts in bacterial adaptation to oxygen deficiency. RegA down-regulated genes involved in DNA replication, cellular division cell envelope biogenesis as well as certain genes in energy metabolism suggesting its impact on bacterial multiplication and adaptation to hypoxia as it enters into the persistence phase. RegA was also found to down-regulate virulence factors such as the virB operon as well as its regulator VjbR. Moreover, this study evaluated the role of two genes BR1614 and BR1510 regulated by RegA and found implicated in fatty acid metabolism. In vivo experiments in mice demonstrated that both genes are required for bacterial survival, multiplication and persistence. In conclusion, RegA was found to regulate, directly and indirectly, the expression of genes that encode for functions in translation, general transcription, energy production and conversion, repair of DNA and protein which represent its high importance and major role in bacterial persistence during brucellosis. 12% of the genome of B. suis is under the control of RegA which makes it a global regulator such as his homologue PrrA in Rhodobacter sphearoides.

La brucellose est une zoonose causée par le genre bactérien Brucella (B.) dont l’incidence mondiale est estimée à 500 000 cas humains par an. Le réservoir est animal, touchant principalement les espèces de rente. Les espèces les plus importantes pour l’Homme sont B. melitensis, B. abortus et B. suis qui partagent plus de 90% d’identité de séquence. Bien qu’elles soient très apparentées sur le plan génétique, elles présentent une diversité de caractéristiques phénotypiques, de préférence d’hôte et de pathogénicité. L’homogénéité génétique de ces espèces peut apparaître comme un atout pour le développement d’outils de diagnostic universels robustes. En revanche, il s’agit d’un challenge pour les distinguer, rendant difficile la caractérisation précise des isolats issus d’un même foyer. Dans le cadre de cette thèse, un outil de diagnostic moléculaire de PCR en temps réel ciblant le genre Brucella a été développé et optimisé. L’outil a été évalué sur des prélèvements de lait de ruminants, ces prélèvements peuvent être une source importante de Brucella et peuvent être utiles au dépistage de la maladie à l’échelle du troupeau. Basée sur la détection de l’élément d’insertion IS711, une séquence présente en plusieurs exemplaires dans le génome, cette méthode affiche des valeurs de sensibilité et de spécificité qui la rendent intéressante pour un schéma global de lutte contre la brucellose. D’autre part, en vue d’améliorer la compréhension de la stabilité génétique de B. melitensis, un panel original de souches isolées dans le cadre d’un foyer et impliquant 4 espèces d’hôtes différentes a été comparé. Ainsi à l’aide de différentes approches complémentaires, leurs séquences génomiques, les caractères phénotypiques ainsi que leurs comportements dans un modèle in vitro ont été comparés. Nos résultats n’ont pas mis en évidence marqueurs qui laisserait à penser que des mutations dans le génome soient indispensables pour s’adapter à un nouvel hôte / Brucellosis is a zoonotic disease caused by the bacterial genus Brucella (B.), whose global incidence is estimated at 500,000 human cases per year. The reservoir is animal, affecting mainly livestock. The most important species for humans are B. melitensis, B. abortus and B. suis, which share more than 90% sequence identity. Although highly genetically related, Brucella spp. exhibit a variety of phenotypic characteristics, host preference and pathogenicity. The genetic homogeneity of these species may appear as an asset for the development of robust universal diagnostic tools. On the other hand, it is a challenge to distinguish them, making it difficult to precisely characterize isolates from the same outbreak. As part of this thesis, a real-time PCR molecular diagnostic tool targeting the genus Brucella was developed and optimized. The method has been evaluated on ruminant milk samples; these samples may be an important source of Brucella and may be useful for herd-scale disease screening. Based on the detection of the IS711 insertion element, a sequence present in several copies within the genome, this method displays sensitivity and specificity values that make it interesting for a global scheme to fight against brucellosis. On the other hand, in order to improve the understanding of the genetic stability of B. melitensis, an original panel of strains isolated in an outbreak and involving four different host species was compared. Thus, using different complementary approaches, their genomic sequences, phenotypic characteristics and their behavior in an in vitro model were compared. Our results did not highlight markers that would suggest that mutations in the genome are essential to adapt to a new host