A Brucella canis é a bactéria responsável pela brucelose nos cães, pode ser transmitida para os seres humanos, ocasionalmente resultando em doença grave, com impacto na saúde pública. A brucelose canina desencadeia inúmeras perdas econômicas em canis comerciais, com a ocorrência de abortamentos, morte embrionária, natimortos e nascimento de filhotes debilitados. O diagnóstico sorológico é rotineiramente realizado, contudo a hemocultura é o teste \"padrão-ouro\". A técnica de reação em cadeia pela polimerase (PCR) pode ser aplicada no diagnóstico direto como alternativa à hemocultura, pela rapidez, alta especificidade e sensibilidade do teste, mas apresenta alto custo com infraestrutura e equipamentos. A amplificação isotérmica mediada por loop (LAMP) constitui outra alternativa para amplificação do DNA em um curto período de tempo, com simplicidade e menor custo. O projeto avaliou comparativamente o desempenho dos testes moleculares de PCR e LAMP com primers direcionados à sequência de inserção IS711 de Brucella spp. em 98 amostras de sangue total, obtidas de 57 cães. Os 57 cães foram divididos em três grupos: infectados por B. canis (cães positivos na hemocultura) não infectados por B. canis (negativos na hemocultura e sem evidências clínicas e epidemiológicas de brucelose) e suspeitos de brucelose (cães negativos na hemocultura, mas com suspeita clínica e/ou epidemiológica da infecção). A sensibilidade e especificidade diagnóstica das reações de LAMP e PCR foram calculadas, utilizando-se os grupos de cães infectados e não infectados, respectivamente. O desempenho dos testes foi analisado, utilizando-se as 98 amostras, comparadas duas a duas, pelos testes estatísticos de Coeficiente Kappa e McNemar. A proporção de amostras positivas foi de 43,87% (43/98) na hemocultura, 46,93% (46/98) na PCR e 16,33% (16/98) na LAMP. A concordância entre a hemocultura e a PCR foi ótima, enquanto que a concordância entre a LAMP e a hemocultura e entre a LAMP e a PCR foi sofrível. A sensibilidade diagnóstica foi de 100% (18/18) na PCR e 44,44% (8/18) na LAMP, enquanto que a especificidade diagnóstica foi de 96% (20/21) na PCR e 100% (21/21) na LAMP. O desempenho da reação de LAMP foi insatisfatório para o diagnóstico da brucelose nos cães, em razão dos baixos valores de sensibilidade do teste. A PCR, por outro lado, apresentou desempenho similar à hemocultura, o que a torna uma alternativa para uso no diagnóstico da brucelose canina. / Brucella canis is the etiological agent responsible for brucellosis in dogs and can be transmitted to human beings, occasionally resulting in severe disease, and leading to impacts on public health. Canine brucellosis triggers numerous economic losses in commercial kennels, causing abortions, embryonic death, stillbirths and birth of debilitated puppies. Serological diagnosis is routinely performed, but blood culture is the gold standard test. Polymerase chain reaction (PCR) can be used to the direct diagnosis of infection in view of its speed and high specificity and sensitivity values, however it has high cost because of the laboratory infrastructure and equipments needed. The loop-mediated isothermal amplification (LAMP) may be an alternative to DNA amplification in a shorter period of time, with simplicity and low cost. This project evaluated the potential of the molecular tests of PCR and LAMP using primers targeting the insertion sequence IS711 of Brucella, using 98 whole blood samples of 57 dogs. The 57 dogs were divided into three groups: infected by B. canis (dogs with positive results in blood culture), non-infected by B. canis (dogs with negative results by blood culture and showing no clinical or epidemiological evidences of brucellosis) and dogs suspected of brucellosis (those with negative blood culture but with clinical and/or epidemiological evidences of infection). The diagnostic sensitivity and specificity of PCR and LAMP were calculated using the infected and non-infected groups, respectively. The performance of the three diagnostic tests was pair compared using the 98 samples using McNemar test and Kappa coefficient. The proportion of positive samples detected by blood culture, PCR and LAMP was respectively 43.87% (43/98), 46.93% (46/98), and 16.33% (16/98). The concordance between blood culture and PCR was almost perfect, while the concordance between LAMP and blood culture and between LAMP and PCR was fair. The diagnostic sensitivity of PCR and LAMP was, respectively, 100% (18/18) and 44.44% (8/18), while the diagnostic specificity of the tests was 96% (20/21) and 100% (21/21), respectively. LAMP performance was not satisfactory for canine brucellosis diagnosis because of the low sensitivity of the test. PCR showed similar performance when compared to blood culture, which makes it a good alternative for use for the diagnosis of canine brucellosis.

A Brucella canis é a bactéria responsável pela brucelose nos cães, pode ser transmitida para os seres humanos, ocasionalmente resultando em doença grave, com impacto na saúde pública. A brucelose canina desencadeia inúmeras perdas econômicas em canis comerciais, com a ocorrência de abortamentos, morte embrionária, natimortos e nascimento de filhotes debilitados. O diagnóstico sorológico é rotineiramente realizado, contudo a hemocultura é o teste \"padrão-ouro\". A técnica de reação em cadeia pela polimerase (PCR) pode ser aplicada no diagnóstico direto como alternativa à hemocultura, pela rapidez, alta especificidade e sensibilidade do teste, mas apresenta alto custo com infraestrutura e equipamentos. A amplificação isotérmica mediada por loop (LAMP) constitui outra alternativa para amplificação do DNA em um curto período de tempo, com simplicidade e menor custo. O projeto avaliou comparativamente o desempenho dos testes moleculares de PCR e LAMP com primers direcionados à sequência de inserção IS711 de Brucella spp. em 98 amostras de sangue total, obtidas de 57 cães. Os 57 cães foram divididos em três grupos: infectados por B. canis (cães positivos na hemocultura) não infectados por B. canis (negativos na hemocultura e sem evidências clínicas e epidemiológicas de brucelose) e suspeitos de brucelose (cães negativos na hemocultura, mas com suspeita clínica e/ou epidemiológica da infecção). A sensibilidade e especificidade diagnóstica das reações de LAMP e PCR foram calculadas, utilizando-se os grupos de cães infectados e não infectados, respectivamente. O desempenho dos testes foi analisado, utilizando-se as 98 amostras, comparadas duas a duas, pelos testes estatísticos de Coeficiente Kappa e McNemar. A proporção de amostras positivas foi de 43,87% (43/98) na hemocultura, 46,93% (46/98) na PCR e 16,33% (16/98) na LAMP. A concordância entre a hemocultura e a PCR foi ótima, enquanto que a concordância entre a LAMP e a hemocultura e entre a LAMP e a PCR foi sofrível. A sensibilidade diagnóstica foi de 100% (18/18) na PCR e 44,44% (8/18) na LAMP, enquanto que a especificidade diagnóstica foi de 96% (20/21) na PCR e 100% (21/21) na LAMP. O desempenho da reação de LAMP foi insatisfatório para o diagnóstico da brucelose nos cães, em razão dos baixos valores de sensibilidade do teste. A PCR, por outro lado, apresentou desempenho similar à hemocultura, o que a torna uma alternativa para uso no diagnóstico da brucelose canina. / Brucella canis is the etiological agent responsible for brucellosis in dogs and can be transmitted to human beings, occasionally resulting in severe disease, and leading to impacts on public health. Canine brucellosis triggers numerous economic losses in commercial kennels, causing abortions, embryonic death, stillbirths and birth of debilitated puppies. Serological diagnosis is routinely performed, but blood culture is the gold standard test. Polymerase chain reaction (PCR) can be used to the direct diagnosis of infection in view of its speed and high specificity and sensitivity values, however it has high cost because of the laboratory infrastructure and equipments needed. The loop-mediated isothermal amplification (LAMP) may be an alternative to DNA amplification in a shorter period of time, with simplicity and low cost. This project evaluated the potential of the molecular tests of PCR and LAMP using primers targeting the insertion sequence IS711 of Brucella, using 98 whole blood samples of 57 dogs. The 57 dogs were divided into three groups: infected by B. canis (dogs with positive results in blood culture), non-infected by B. canis (dogs with negative results by blood culture and showing no clinical or epidemiological evidences of brucellosis) and dogs suspected of brucellosis (those with negative blood culture but with clinical and/or epidemiological evidences of infection). The diagnostic sensitivity and specificity of PCR and LAMP were calculated using the infected and non-infected groups, respectively. The performance of the three diagnostic tests was pair compared using the 98 samples using McNemar test and Kappa coefficient. The proportion of positive samples detected by blood culture, PCR and LAMP was respectively 43.87% (43/98), 46.93% (46/98), and 16.33% (16/98). The concordance between blood culture and PCR was almost perfect, while the concordance between LAMP and blood culture and between LAMP and PCR was fair. The diagnostic sensitivity of PCR and LAMP was, respectively, 100% (18/18) and 44.44% (8/18), while the diagnostic specificity of the tests was 96% (20/21) and 100% (21/21), respectively. LAMP performance was not satisfactory for canine brucellosis diagnosis because of the low sensitivity of the test. PCR showed similar performance when compared to blood culture, which makes it a good alternative for use for the diagnosis of canine brucellosis.

Made available in DSpace on 2016-08-15T20:31:15Z (GMT). No. of bitstreams: 1 KeniaSMA_DISSERT.pdf: 1150269 bytes, checksum: 54d277c76ed42d8d5e0470e502ceca2f (MD5) Previous issue date: 2012-08-31 / This study was aimed at researching anti-Leptospira spp. and anti-Brucella spp. agglutinins in cattle slaughtered in public slaughterhouses in the State of Rio Grande do Norte, as well as confirm the presence of these agents through PCR in the ovaries and epididymides of the slaughtered animals. For such purpose, serum, and ovary or epididymides were collected from cattle at the public slaughterhouses of that state. Sampling was performed during normal slaughter line, without any interference as the order, sex, age, or origin of the slaughtered animals. The screening of animal reagent to leptospirosis was made through the Microscopic Agglutination Test (MAT). To search for brucellosis, animals were screened by the Buffered Acidified Antigen test (BAA) and as a confirmatory test 2-mercaptoethanol (2-ME) was used. To detect bacterial DNA in samples of ovary and epididymides the Polymerase Chain Reaction (PCR) was used. Of all the 306 animals evaluated, 189 (61.76%) were positive for the MAT, where the predominant serovar was Hardjo (24.9%). The second most frequent serovar was Wolffi (14.8%), followed by Butembo, Icterhaemorrragiae, and Hebdomadis who contributed with frequencies ranging from 6.9% to 2.6%. Lower frequencies were found for the serovars Australis (1.6%), Pomona (1.1%), Grippotyphosa (1.1%) and Patoc (1.1%). Canicola, Autumnalis, and Sentot had frequencies lower than 1%. Three animals were positive for the BAA test, however only one of these reacted positively to 2-ME. Not one of the samples was positive for Brucella spp. and Leptopira spp. DNA through the PCR technique. Given the above mentioned data it was concluded that the Leptospira spp. infection in cattle in the semiarid region of Rio Grande do Norte occurs at higher frequency with the serovar Hardjo during the dry season of the year. The Brucella spp. infection is present but has very low frequency, and no DNA from Leptospira spp. and Brucella spp. was detected in the ovary and epididymis samples of the cattle examined / Objetivou-se pesquisar aglutininas anti-Leptospira spp. e anti-Brucella spp. em bovinos de abatedouros públicos no Estado do Rio Grande do Norte, bem como confirmar através da PCR, a presença desses agentes nos ovários e epidídimos desses animais. Para isso, foram colhidos durante a linha normal de abate, o sangue e ovário ou epidídimo, de 306 animais no período de dezembro de 2010 a agosto de 2011. Através da Reação de Soroaglutinação Microscópica (SAM) foram detectados 189 (61,76%) animais sororeagente para Leptospira spp., sendo o sorovar Hardjo (24,9%) o mais predominante, seguido do Wolffi, Butembo, Icterhaemorrragiae e Hebdomadis que contribuiram com frequências que variaram de 14,8% a 2,6%. Os sorovares Australis, Pomona, Grippotyphosa, Patoc, Canícola, Autumnalis e Sentot foram menos frequentes. Em análise em separado, o sorovar Hadjo apresentou maior ocorrência no período seco do que no chuvoso (p=0,031). Para a brucelose os animais foram triados através da prova do Antígeno Acidificado Tamponado (AAT) e confirmados pela prova do 2-mercaptoetanol (2-ME). Três animais foram positivos ao AAT, desses apenas um reagiu positivamente ao 2-ME. A PCR foi utilizada para detecção do DNA bacteriano nas amostras de ovário e epidídimo. Nenhuma amostra foi positiva na PCR para detecção de DNA da Brucella spp. e Leptospira spp. Diante do exposto conclui-se que a infecção por Leptospira spp. em bovinos ocorre no semiárido do Rio Grande do Norte com maior frequência para o sorovar Hardjo durante o período seco do ano. A brucelose foi detectada, mas com baixa frequência. Através da PCR não foi detectado DNA de Leptospira spp. e Brucella spp. nos ovários e epidídimos dos animais

Brucella est une bactérie intracellulaire facultative à Gram négatif responsable d’une zoonose, la brucellose. Pour persister dans l’organisme, Brucella agit comme un pathogène furtif en modulant la réponse immunitaire de l’hôte. La cyclooxygénase 2 (COX-2) est l’enzyme responsable de la synthèse des prostanoïdes, des médiateurs lipidiques dérivés de l’acide arachidonique (AA) présentant des propriétés immunorégulatrices. Cette thèse est centrée sur l’étude de cette voie métabolique au cours de l’infection par Brucella in vitro dans des cellules dendritiques (DC) humaines et murines ainsi qu’in vivo chez la souris en comparant différentes routes d’infection. Nous avons mis en évidence la capacité de l’infection à stimuler in vitro la production d’AA ainsi que l’expression de Ptgs2. In vivo, la comparaison des différentes routes d’inoculation a montré que l’infection intradermale induit une signature génique inflammatoire caractérisée par l’expression de Ptgs2 et d’Ifng. L’utilisation de NS-398, un inhibiteur spécifique de COX-2 stimule la clairance bactérienne dans les ganglions cervicaux (CLN) drainant le site d’infection. Ces résultats ouvrent ainsi la voie à de nouvelles stratégies thérapeutiques dans le traitement de la brucellose. La seconde partie de la thèse traite de l’implication des infections bactériennes dans l’initiation des processus oncogéniques. Nous y présentons une revue répertoriant l’ensemble des mécanismes pouvant contribuer à l’initiation oncogénique ainsi qu’un projet que nous développons au laboratoire portant sur l’initiation d’un lymphome folliculaire à la suite d’une stimulation antigénique chronique suite à l’infection par B. abortus. / Brucella is a facultative intracellular Gram-negative bacterium, responsible for a zoonosis called brucellosis. To persist into the host, Brucella acts as a stealthy pathogen by modulating the host immune response. The cyclooxygenase 2 (COX-2) is the enzyme responsible for the synthesis of prostanoids, a family of lipid mediators derived from the arachidonic acid (AA) and presenting immunomodulatory properties. Here, we have studied the impact of this pathway during Brucella infection in vitro in human and murine dendritic cells (DCs) as well as in vivo by comparing different infection routes. We have highlighted the ability of the infection to stimulate the AA synthesis and Ptgs2 expression. In vivo, by comparing different inoculation routes we showed that intradermal infection induces a specific inflammatory gene signature characterized by an important expression of Ptgs2 and Ifng. The use of NS-398, a specific inhibitor of COX-2 stimulates the bacterial clearance in the cervical lymph nodes (CLN) draining the site of infection. These results might open the way to new therapeutic strategies in the treatment of brucellosis. In a second part of the thesis, we discuss the involvement of bacterial infections in initiating oncogenic processes. Here, we present a review listing all the mechanisms that contribute to the oncogenic initiation and a project that we are developing in the laboratory dealing with the initiation of follicular lymphoma following chronic antigenic stimulation during B. abortus infection.

L'accentuation des phénomènes de résistance aux antibiotiques augmente la difficulté d'enrayer les infections bactériennes. Afin de palier ce problème, la mise au point de nouvelles stratégies, telle la stratégie antivirulence, est essentielle. Ainsi, ce manuscrit propose une nouvelle approche thérapeutique contre les bactéries à développement intracellulaire. Les analyses génomiques ont permis de mettre en évidence l'histidinol déshydrogénase (HDH, EC 1.1.1.23), enzyme impliquée dans la biosynthèse de l'histidine, comme cible biologique pour la conception de nouveaux agents antibactériens. L'étude de l'inhibition de cette dernière permet une validation de l'approche sur Brucella suis, agent responsable de la brucellose, et un élargissement du spectre d'action des composés mis au point est envisagé par l'inhibition de HDH de Mycobacterium tuberculosis. Les travaux préliminaires nécessaires a cet élargissement sont présentés dans ce manuscrit. / The raise of antibiotic resistances increases the difficulty to eradicate bacterial infections. The development of new therapeutic approaches, such as the antivirulence strategy, is essential to limitate the impact of this phenomenon. This manuscript details a new therapeutic approach against intracellular pathogens. Genomic analyses allowed to discover new targets. The histidinol dehydrogenase (HDH, EC 1.1.1.23), which is an enzyme involved in histidine biosynthesis, has therefore be chosen for the conception of new antibacterial compounds. Inhibition studies of HDH of Brucella suis allows a validation of the strategy. In order to confirm the width of the therapeutic spectrum of synthesised compounds, the inhibition of HDH from Mycobacterium tuberculosis is envisaged and preliminary experiments are presented.

Objetivos: Conocer la prevalencia de anticuerpos contra Brucella sp en donantes de sangre de la Unidad de Medicina Transfusional del Hospital Edgardo Rebagliatti Martins. Diseño: Estudio descriptivo, prospectivo de corte transversal. Materiales y métodos: Se analizaron las muestras de 1300 donantes de sangre. Como prueba de tamizaje para la detección de anticuerpos anti-Brucella se aplico la prueba de Rosa de Bengala; las muestras positivas fueron evaluadas por las pruebas de aglutinación en tubo (AT) y 2-Mercaptoetanol(2-ME), respectivamente. Resultados: Dos donantes (0.19%), fueron positivos a la prueba Rosa de Bengala, resultados confirmados por las pruebas AT (títulos de 100 y 50 respectivamente) y 2-ME (títulos de 50 y 25 respectivamente). Conclusiones: Se demostró la presencia de donantes con posible enfermedad activa, confirmada mediante AT y 2-ME, dentro de la población de donantes del hospital, evidenciándose la posibilidad de transmisión de la enfermedad. Se deben realizar mayores estudios con la finalidad de conocer la realidad de otros bancos de sangre.

Memoria para optar al Título Profesional de Médico Veterinario / La Brucelosis bovina es una enfermedad que ha llevado a los distintos países a establecer estrategias de control y prevención de la enfermedad. En Chile las estrategias establecidas por el Servicio Agrícola y Ganadero (SAG) son vigilancia, saneamiento y prevención en el ganado bovino. Esta última medida consiste en la vacunación con B. abortus cepa RB51. La ventaja de esta cepa es que no produce interferencia diagnóstica con pruebas que utilizan el lipopolisacárido liso (s-LPS) como antígeno. El presente estudio caracterizó la respuesta inmune humoral y celular generada en el ganado bovino vacunado con esta cepa. Para esto se contó con un grupo de 25 terneras las cuales fueron vacunadas con 1- 3 x 1010 UFC de B. abortus RB51, entre la edad de 4 y 8 meses. Estas fueron mantenidas y manejadas en su rebaño durante el período de estudio en el cual se realizaron cuatro muestreos los días 0, 30, 180 y 360 post vacunación, recolectando dos muestras de sangre de cada animal una con y otra sin anticoagulante (heparina de sodio). Las muestras obtenidas fueron procesadas en cinco pruebas diagnósticas distintas y éstas son: rosa de bengala, ELISA indirecto, ELISA competencia las cuales detectan la respuesta inmune humoral al s-LPS de Brucella. También se usaron la prueba de ELISA citosol de RB51 que detecta la respuesta inmune humoral frente a proteínas del citosol de RB51 y finalmente la prueba de IFN bovino, que detecta la respuesta inmune celular frente a RB51. Los resultados obtenidos, demostraron que la cepa RB51 no induce anticuerpos contra el s-LPS de Brucella tal como era esperado, pero sí induce anticuerpos frente a proteínas citosólicas de cepa RB51. Durante el período de estudio, sólo una vaquilla arrojó resultados positivos a estas pruebas y esto se debió probablemente al resultado de una infección natural como consecuencia de la presencia de brucelosis en el rebaño. Los resultados obtenidos frente a la prueba de IFN bovino demostraron que cepa RB51 induce respuesta inmune celular, detectable mediante la presencia de esta citoquina que fue liberada al plasma bovino por los linfocitos estimulados con el citosol de RB51. La evolución del porcentaje de animales negativos a las pruebas que usan s-LPS como antígeno fue estable ya que, exceptuando la vaquilla positiva antes mencionada, todos los animales permanecieron negativos a los cuatro muestreos. La prueba de ELISA citosol de RB51muestra un evidente aumento en el porcentaje de animales positivos entre el primer y segundo muestreo, para luego permanecer constante en los muestreos siguientes, esto debido a los anticuerpos generados como respuesta post vacunatoria a las proteínas de RB51, que decaen con el tiempo. Finalmente la prueba de IFN bovino muestra un aumento del porcentaje de animales positivos entre el primero y segundo muestreo y luego decrece en forma constante, hasta el último muestreo / Financiamiento: IFS B/1800-2

Memoria para optar al Título Profesional de Médico Veterinario / La Brucelosis canina es una enfermedad infectocontagiosa zoonótica causada por la bacteria Brucella canis, afectando principalmente el sistema reproductivo de los perros en ambos sexos. Los signos de esta enfermedad no son evidentes clínicamente e incluso muchos pueden ser asintomáticos. Se caracteriza por producir aborto tardío en hembras y orquitis, epididimitis e infertilidad en machos, pero muchas veces estos signos pueden pasar desapercibidos. En este estudio se determina la sensibilidad analítica de un ensayo PCR tanto para muestras de sangre contaminadas como en cultivos puros para el diagnóstico de brucelosis canina, comparando dos métodos de extracción de DNA. Como resultado se obtuvo que la prueba PCR es capaz de detectar la bacteria en sangre pero obteniendo resultados positivos sólo con uno de los métodos de extracción, detectándose DNA hasta el nivel de los femtogramos / Proyecto FIV 121014019102003

RESUME : Le Quorum Sensing est un système de communication bactérien permettant à une population de coordonner l’expression de gènes cibles en fonction de sa densité ou des propriétés du milieu (diffusion, flux....). Chez les bactéries à Gram négatif, le Quorum Sensing est basé sur la synthèse et la détection de petites molécules signal appelées N-acyl-homosérine lactones (AHLs). Les régulateurs transcriptionnels de type LuxR sont les médiateurs de ce système de régulation. Lorsque la concentration en AHLs augmente, ces molécules se fixent au domaine N-terminal d’un régulateur LuxR et provoquent des changement conformationnels entraînant une modification de l’activité du régulateur. Un tel système de régulation a été mis en évidence chez la bactérie Gram négative Brucella melitensis. Cette bactérie pathogène intracellulaire synthétise une dodécanoyl-homosérine lactone (C12-HSL) et possède deux régulateurs de type LuxR : VjbR et BabR. VjbR est impliqué dans la virulence de B. melitensis et est indispensable à l’expression de deux facteurs de virulence: le système flagellaire et le système de sécrétion de type IV VirB. Les C12-HSL ont quant à elles un effet répresseur sur ces deux structures membranaires. Durant ce travail, la mutation du domaine Nterminal du régulateur VjbR a permis de démontrer la capacité de VjbR à médier l’effet des C12-HSL sur l’opéron virB. Les souches mutées dans le gène vjbR forment des agrégats en cultures liquides. Nous avons montré que ce phénotype est lié à la production d’un exopolysaccharide, suggérant pour la première fois que Brucella pourrait former des structures de type biofilm. Cette étude a également permis de mettre en évidence un rôle majeur de VjbR dans la régulation de structures de surface puisque ce régulateur est impliqué dans le contrôle de l’expression de nombreuses protéines de membrane externe (Omp). L’utilisation de la technique d’immunoprécipitation chromatinienne (ChIP) a permis de montrer que VjbR régule directement deux de ces Omps ainsi que l’opéron virB. La virulence de Brucella est en partie basée sur sa capacité à dévier le trafic intracellulaire de ses cellules hôtes (phagocytes professionnels et nonprofessionnels) et à s’y multiplier. Lors de son cycle infectieux, Brucella est confrontée à de nombreux stress et environnements différents, suggérant la nécessité d’une régulation génétique fine en réponse à des stimuli environnementaux. Le Quorum Sensing, de par son implication dans la virulence de ce pathogène pourrait être impliqué dans de telles régulations. Afin d’aborder de façon globale le rôle de VjbR et de BabR chez B. melitensis, des études transcriptomique et protéomique des mutants ΔvjbR et ΔbabR ont été réalisées. Ces études ont permis de mettre en évidence que le Quorum Sensing chez B. melitensis est un système de régulation global, puisqu’il permet de réguler 10% du génome dans les conditions testées (dont 9% sous le contrôle de VjbR). De nombreuses cibles de ces régulateurs sont impliquées dans la virulence et l’adaptation aux conditions de stress (oxydatif, métabolique...), suggérant un rôle important du Quorum Sensing dans l’accomplissement du cycle infectieux de B. melitensis. SUMMARY : Quorum Sensing is a bacterial communication system wich allows the coordinated gene expression within a population regarding its density and environmental properties (diffusion, flow...). In Gram negative bacteria, Quorum Sensing is based on the synthesis and the detection of small diffusible molecules called N-acyl-homoserine lactones (AHLs). LuxR transcriptional regulators are the mediators of these regulation systems. When AHL concentration increases, these molecules bind to the N-terminal domain of a LuxR-type regulator and leads to conformational changes driving the modification of the regulator activity. A similar regulation system has been discovered in the Gram negative bacteria Brucella melitensis. This intracellular pathogen synthesizes a dodecanoylhomoserine lactone (C12-HSL) and possesses two LuxR-type regulators: VjbR and BabR. VjbR is involved in the virulence of this pathogen and is crucial for the expression of two virulence factors : the flagellar system and the type four secretion system VirB. C12-HSL have a repressor effect on these two membrane structures. During this work, mutation of the N-terminal domain of VjbR allowed us to demonstrate the ability of VjbR to mediate C12-HSL effect on the virB operon. vjbR mutated strains aggregate in liquid cultures. We have demonstrated that this phenotype is linked to the production of an exopolysaccharide, suggesting for the first time that Brucella could form biofilm-type structures. This study also demonstrates that VjbR has a major role in the regulation of surface structures because this regulator controls the expression of many outer membrane proteins (Omp). Using the chromatin immunoprecipitation technique (ChIP), we have shown that two of these Omps, as well as the virB operon, are directly regulated by VjbR. The virulence of Brucella is partly based on its ability to deviate the intracellular traffic of its host cells (professional and nonprofessional phagocytes) and to proliferate within these cells. During its infectious cycle, Brucella faces numerous stresses and environments, suggesting the necessity of a finely tuned genetic regulation depending on environmental stimuli. Quorum Sensing, through its involvement in the virulence of this intracellular pathogen, could be involved in such regulations. In order to investigate the role of VjbR and BabR in B. melitensis, global transcriptomic and proteomic studies of ΔvjbR and ΔbabR mutants were performed. These studies demonstrate that Quorum Sensing is a global regulation system in B. melitensis because it controls the expression of 10% of the genome in the condition tested (9% through VjbR). Numerous targets of these two regulators are involved in virulence and adaptation to environmental stresses (oxydative, metabolic...), suggesting an important role of Quorum sensing in the achievement of the infectious cycle of B. melitensis.

Un brote de bovinos de raza Hereford fueron inoculados a repetición con Brucella abortus cepa 19 durante un período de doce meses. Elaboraron anticuerpos correspondientes a la clase Ig M e Ig G, siendo los últimos los que predominaron y persistieron hasta el final del experimento. Los anticuerpos de la clase Ig G correspondieron a poblaciones de anticuerpos diferentes: aglutinantes y no aglutinantes o bloqueantes, estos detectados por reacción de Coombs. Ambos tipos de anticuerpos fueron separados mediante inmunoadsorciones seriadas y purificadas por cromatografía de intercambio iónico. Se les caracterizó como pertenecientes aa calse Ig G1, siendo estudiadas sus propiedades inmunoquímicas y biológicas. Los anticuerpos aglutinantes fueron capaces de aglutinar el antígeno, fijar complemento y facilitar la depuración sanguínea de Brucella abortus cepa 19 marcada con 131 I en ratones e inhibieron la multiplicación de Brucella abortus en el bazo de ratones inoculados con estas bacterias. Por el contrario los anticuerpos coaglutinantes, fueron incapaces de facilitar la depuración sanguínea de bacterias y facilitaron su multiplicación en bazo. A la luz de estos resultados postulamos la hipótesis de que estos anticuerpos intervendrían en el facilitamiento de la permanencia y multiplicación de las brucelas en el huésped, siendo éste, uno de los mecanismos responsables de la iniciación y/o mantenimiento de la cronicidad en la brucelosis bovina. / Hereford cattles were repeatedely inoculated with Brucella abortus strain 19 for twelve months. Antibodies elaborated belonging to classes Ig M and Ig G, thelatter being the ones that predominated and persisted till the end of the experiment. Ig G antibodies were related to two different types of antibodies: agglutinating and non-agglutinating antibodies, were specifically purified by immunoadsortion and latter purified by ionic exchange chromatography. They were characterised as Ig G1, and its inmunochemical and biological properties were studied. Agglutinating antibodies were able to agglutinate the antigen, fix the complement and increased blood clearance of Brucella abortus strain 19 marked with 131 I in mice and inhibited the multiplication of Brucella in mice spleen inoculated with these bacteria. On the other hand, the co-agglutinating antibodies unable to facilate the bacterial blood clearence and enhaced its multiplication in spleen. Owing to these results we postulate that these antibodies would facilitate the permanence and multiplication of bacteria in the guest. This mechanism would be responsible for the beginning and maintainans of chronic cattle brucellosis.