Brucellosis is a zoonotic disease caused by a Gram-negative coccobacillus. There are four Brucella strains of zoonotic importance in our domestic species, subdivided by their culture phenotypes: Brucella abortus (B. abortus, B. melitensis, B. suis (smooth strains) and B. canis (rough strain). Dogs can serve as hosts for all four of the zoonotic strains; however, routine serologic testing in dogs has been limited to the identification of antibodies to B. canis. The aim of our study was to identify a serologic test that can be utilized to identify smooth Brucella strain antibodies in canines. We hypothesize that the Brucella abortus Fluorescence Polarization Assay would be successful in identifying antibodies to smooth Brucella strain in canines. Ninety-five dogs, including forty-five hog hunting dogs were screened for circulating antibodies to any of the four zoonotic strains of the bacteria utilizing a combination of Canine Brucella Slide Agglutination Test (CBSA), Brucella canis Agar Gel Immunodiffusion II test (AGID), Brucella abortus Card Agglutination Test (BCA), and the Brucella abortus Fluorescence Polarization Assay (FPA). Test interpretation results yielded a 0% (0/95) smooth Brucella strain seropositivity rate, with 2% (2/95) of dogs yielding inconclusive rough Brucella strain serology results (0-2% rough strain seropositivity rate). Additionally, a retrospective portion of the study was performed to identify sera containing circulating antibodies to any of the smooth strains of Brucella by testing previously banked canine serum samples stored at Cornell's Veterinary Diagnostic Laboratory from 2018-2019 via Brucella abortus FPA. Of the 769 serum samples tested, 13/769 (1.7%) yielded an inconclusive result, 725/769 (94.2%) were negative, 30/769 (4%) yielded a positive FPA test result, and 1/769 (0.1%) had to be excluded due to insufficient sample remaining to perform the diagnostic test. Of the 30 FPA positive canine serum samples, 97% (29/30) also tested positive on the CBSA test. Additionally, there was a statistically significant (p <0.0001) likelihood of altered (spayed/neutered) and mixed breed dogs to be FPA positive when compared to intact, purebred dogs. / Master of Science / Brucellosis is a bacterial disease that can cause severe reproductive, orthopedic and general illness in both dogs and humans. There are four different species of Brucella that can be transmitted between animals and people: Brucella canis, abortus, suis, and melitensis. Although Brucella canis is the species that is widely recognized and breeding dogs are routinely tested for this strain, we have vastly under recognized the ability for dogs to contract and transmit the other three (smooth) Brucella species. Of added concern is the fact that the test currently used to screen dogs for brucellosis only identifies Brucella canis infection. Thus, veterinarians may be missing cases where dogs are infected with other Brucella species. This study revealed promising evidence in identification of smooth Brucella strain antibodies in canines, particularly altered and mixed breed canines, via the Brucella abortus Fluorescent Polarization Assay. The contributions of this study are threefold. First, to heighten awareness that both smooth and rough strains of brucellosis infection in dogs are infectious diseases of zoonotic concern. Second, it demonstrates that smooth Brucella strain infection along with the traditional strategy of selectively screening dogs breeding dogs may be underestimating the prevalence of brucellosis among the canine population in the United States thus, supporting the need for broadened screening recommendations. Third, it reveals the need for a commercially-available, validated test for the smooth strains of brucellosis in dogs and offers direction for future research efforts to likely focus on the validation of the Brucella abortus Fluorescent Polarization Assay.

Bacterias patógenas como Salmonella, habitantes naturales del tracto intestinal de diversos animales incluido el hombre, están comúnmente presentes en efluentes de desagües principalmente domésticos que desembocan al mar en forma directa o indirecta a través de ríos y acequias, llegando a constituir parte de la flora contaminante del litoral limeño y de los alimentos provenientes del mismo. La presencia de Salmonella está determinada por las condiciones biológicas y fisicoquímicas del ambiente acuático, que permiten su supervivencia, desarrollando mecanismos de adaptación como entrar a un estado de “viable no cultivable”, cambios metabólicos o alteraciones genómicas en respuesta a condiciones ambientales adversas. Debido a la importancia que tiene Salmonella en la incidencia de enfermedades gastrointestinales, se hace necesaria su vigilancia en el medio ambiente acuático para lo cual es preciso desarrollar estudios que faciliten el aislamiento, identificación y diferenciación de estirpes patogénicas en ambientes naturales. Muestras de agua de mar tomadas a lo largo del litoral limeño, durante los meses de marzo, abril y mayo del 2000, fueron procesadas para el aislamiento de Salmonella usando el método de filtración en membrana, pre-enriquecimiento en Agua Bufferada Peptonada; enriquecimiento selectivo en caldo Tetrationato, Selenito Cistina y Rappaport-Vassiliadis y posterior aislamiento selectivo en Agar XLD, Sufito de Bismuto, BPLS, SS, Hektoen y XLD. La identificación bioquímica se realizó mediante las pruebas de Oxidasa, Catalasa, Indol, Urea, TSI, LIA, Citrato y RM-VP. Para la identificación serológica se utilizaron sueros polivalentes agrupadores (A, B, C1, C2, D, E1 y E4), suero capsular Vi y flagelares de Salmonella choleraesuis. Se seleccionaron 203 estirpes oxidasa negativos, catalasa positivos, de las que 18 fueron identificadas como Salmonella choleraesuis, de las cuales 10 cepas fueron del serogrupo C1 y serotipo Salmonella Djugu y 8 del serogrupo D y serotipo Salmonella Enteritidis, una con bioquímica típica y 2 cepas atípicas incluidas en el serogrupo B de Salmonella choleraesuis, 3 cepas rugosas y 24 cepas con bioquímica típica que no aglutinaron con el suero polivalente empleado. Los perfiles de proteínas totales obtenidos mediante PAGE-SDS señalan diferencias de las cepas entre e intra serovar luego del análisis estadístico. El ADN cromosómico de los serotipos identificados fueron cortados con endonucleasas de restricción e hibridados con una sonda específica para el gen rDNA16S marcada por quimioluminiscencia. Se encontraron 4 ribotipos (RB, RC1, RC2 y RD), que correspondieron a cada serovar de Salmonella choleraesuis. / --- Pathogenic bacteria like Salmonella, natural inhabitants of the intestinal tract of diverse animals included the man, they are commonly present in effluents of mainly domestic drainages that discharge into the sea in direct form or through rivers and canals, ending up constituting part of the contaminate flora of the Lima´s coast and of the foods coming from the same one. Their presence is determined by the biological and physico chemical conditions of the aquatic environment that allow its survival, developing mechanisms of adaptation like to enter to state of “viable but no culturable”, metabolic and genomic changes in answer to adverse environmental conditions. Due to the importance that Salmonella has in the incidence of gastrointestinal illnesses, it becomes necessary their surveillance in the aquatic environment for that which is necessary to develop studies that facilitate the isolation, identification and differentiation of parthogenic strains in natural ecosystems. Seawater samples were taked along the Lima´s coast, during the months of march, april and may of 2000. For the isolation the membrane filtration method was used, pre-enrichment in Buffered Peptoned Water; selective enrichment in Tetrathionate, Selenite Cystine and Rappaport-Vassiliadis broths and selective isolation in XLD, Bismuth Sulfite, BPLS, SS, Hektoen, XLD agars. In the biochemical identification Oxidasa, Catalasa, Indole tests were used and it was cultivated in Urea, TSI, LIA, Citrato, RM-VP media. For the serologic identification grouping polyvalents (A, B, C1, C2, D, E1 and E4) and flagellars sera of Salmonella choleraesuis were used. 203 strains negative oxidase and positive catalase were selected, of which 18 were identified as Salmonella choleraesuis, inside those that 10 strains belonged to C1 serogroup and Salmonella Djugu serotype, and 8 to D serogroup and Salmonella Enteritidis serotype, one with typical biochemistry and 2 atypical strains typing inside the B serogroup of Salmonella choleraesuis, 3 rough strains and 24 with typical biochemistry that they didn't agglutinate with the polyvalent serum used. The profiles of total proteins obtained by means of SDS-PAGE point out differences of the strains inter and intra serovar after statistical analyses. The chromosomal DNA of the identified serotypes was cut with restriction endonucleases and hibridized with a specific probe for the gene rDNA16S marked by chemioluminiscens. They were 4 ribotypes (RB, RC1, RC2 y RD), that corresponded to each serovar of Salmonella choleraesuis.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Bacterias patógenas como Salmonella, habitantes naturales del tracto intestinal de diversos animales incluido el hombre, están comúnmente presentes en efluentes de desagües principalmente domésticos que desembocan al mar en forma directa o indirecta a través de ríos y acequias, llegando a constituir parte de la flora contaminante del litoral limeño y de los alimentos provenientes del mismo. La presencia de Salmonella está determinada por las condiciones biológicas y fisicoquímicas del ambiente acuático, que permiten su supervivencia, desarrollando mecanismos de adaptación como entrar a un estado de “viable no cultivable”, cambios metabólicos o alteraciones genómicas en respuesta a condiciones ambientales adversas. Debido a la importancia que tiene Salmonella en la incidencia de enfermedades gastrointestinales, se hace necesaria su vigilancia en el medio ambiente acuático para lo cual es preciso desarrollar estudios que faciliten el aislamiento, identificación y diferenciación de estirpes patogénicas en ambientes naturales. Muestras de agua de mar tomadas a lo largo del litoral limeño, durante los meses de marzo, abril y mayo del 2000, fueron procesadas para el aislamiento de Salmonella usando el método de filtración en membrana, pre-enriquecimiento en Agua Bufferada Peptonada; enriquecimiento selectivo en caldo Tetrationato, Selenito Cistina y Rappaport-Vassiliadis y posterior aislamiento selectivo en Agar XLD, Sufito de Bismuto, BPLS, SS, Hektoen y XLD. La identificación bioquímica se realizó mediante las pruebas de Oxidasa, Catalasa, Indol, Urea, TSI, LIA, Citrato y RM-VP. Para la identificación serológica se utilizaron sueros polivalentes agrupadores (A, B, C1, C2, D, E1 y E4), suero capsular Vi y flagelares de Salmonella choleraesuis. Se seleccionaron 203 estirpes oxidasa negativos, catalasa positivos, de las que 18 fueron identificadas como Salmonella choleraesuis, de las cuales 10 cepas fueron del serogrupo C1 y serotipo Salmonella Djugu y 8 del serogrupo D y serotipo Salmonella Enteritidis, una con bioquímica típica y 2 cepas atípicas incluidas en el serogrupo B de Salmonella choleraesuis, 3 cepas rugosas y 24 cepas con bioquímica típica que no aglutinaron con el suero polivalente empleado. Los perfiles de proteínas totales obtenidos mediante PAGE-SDS señalan diferencias de las cepas entre e intra serovar luego del análisis estadístico. El ADN cromosómico de los serotipos identificados fueron cortados con endonucleasas de restricción e hibridados con una sonda específica para el gen rDNA16S marcada por quimioluminiscencia. Se encontraron 4 ribotipos (RB, RC1, RC2 y RD), que correspondieron a cada serovar de Salmonella choleraesuis. / Pathogenic bacteria like Salmonella, natural inhabitants of the intestinal tract of diverse animals included the man, they are commonly present in effluents of mainly domestic drainages that discharge into the sea in direct form or through rivers and canals, ending up constituting part of the contaminate flora of the Lima´s coast and of the foods coming from the same one. Their presence is determined by the biological and physico chemical conditions of the aquatic environment that allow its survival, developing mechanisms of adaptation like to enter to state of “viable but no culturable”, metabolic and genomic changes in answer to adverse environmental conditions. Due to the importance that Salmonella has in the incidence of gastrointestinal illnesses, it becomes necessary their surveillance in the aquatic environment for that which is necessary to develop studies that facilitate the isolation, identification and differentiation of parthogenic strains in natural ecosystems. Seawater samples were taked along the Lima´s coast, during the months of march, april and may of 2000. For the isolation the membrane filtration method was used, pre-enrichment in Buffered Peptoned Water; selective enrichment in Tetrathionate, Selenite Cystine and Rappaport-Vassiliadis broths and selective isolation in XLD, Bismuth Sulfite, BPLS, SS, Hektoen, XLD agars. In the biochemical identification Oxidasa, Catalasa, Indole tests were used and it was cultivated in Urea, TSI, LIA, Citrato, RM-VP media. For the serologic identification grouping polyvalents (A, B, C1, C2, D, E1 and E4) and flagellars sera of Salmonella choleraesuis were used. 203 strains negative oxidase and positive catalase were selected, of which 18 were identified as Salmonella choleraesuis, inside those that 10 strains belonged to C1 serogroup and Salmonella Djugu serotype, and 8 to D serogroup and Salmonella Enteritidis serotype, one with typical biochemistry and 2 atypical strains typing inside the B serogroup of Salmonella choleraesuis, 3 rough strains and 24 with typical biochemistry that they didn't agglutinate with the polyvalent serum used. The profiles of total proteins obtained by means of SDS-PAGE point out differences of the strains inter and intra serovar after statistical analyses. The chromosomal DNA of the identified serotypes was cut with restriction endonucleases and hibridized with a specific probe for the gene rDNA16S marked by chemioluminiscens. They were 4 ribotypes (RB, RC1, RC2 y RD), that corresponded to each serovar of Salmonella choleraesuis.

Submitted by Nathália Faria da Silva (nathaliafsilva.ufv@gmail.com) on 2017-07-13T13:35:59Z No. of bitstreams: 1 texto completo.PDF: 380446 bytes, checksum: 7e55c93cd23967dc3c52fb63f4b4ab1f (MD5) / Made available in DSpace on 2017-07-13T13:35:59Z (GMT). No. of bitstreams: 1 texto completo.PDF: 380446 bytes, checksum: 7e55c93cd23967dc3c52fb63f4b4ab1f (MD5) Previous issue date: 2001-10-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / No presente trabalho, estudaram-se aspectos epidemiológicos das infecções causadas por Streptococcus suis, enfocando, principalmente, a ocorrência dos seus diversos sorotipos. Analisaram-se 323 amostras de S. suis, isoladas de animais clinicamente doentes, as quais foram sorotipadas, de acordo com a técnica de coaglutinação, utilizando soro hiperimune produzido para este trabalho. Um questionário foi elaborado e enviado aos proprietários das granjas questionários recebidos, positivas para observou-se que S. todas suis. as Analisando granjas os possuem sistema de criação de ciclo completo, 40,5% possuem entre 10 e 20 anos de existência e 38,1% possuem entre 100 e 500 matrizes. Pela sorotipagem das amostras, constatou-se que o S. suis está presente em vários estados brasileiros, sendo o maior número de isolados correspondente ao Estado de Minas Gerais (60,1%), seguido de São Paulo (10,4%) e Paraná (8,9%). O sorotipo 2 foi o mais freqüente (61,9%), seguido dos sorotipos 1, 3, 4, 7 e 8. O maior número de isolamentos foi obtido de cérebro (56,0%), seguido do pulmão (9,8%) e casos de septicemia (9,2%). Em relação à sensibilidade a antibióticos, as amostras de S. suis foram mais sensíveis a amoxicilina (96,4%), seguida de lincomicina-espectinomicina (90,9%), ampicilina (87,2%), cloranfenicol (79,1%) e penicilina (71,0%). / In the present work, epidemiological features of Streptococcus infections were studied, mainly focalizing the serotypes suis occurrence. It was analyzed 323 S. suis isolated samples, of clinically sick pigs. These samples were serotyped following the coaglutination method, using hyperimmune sera, specially produced for this work. A questionnaire was developed and then sent to farmers that had S. suis positives pigs. Analyzing the received questionnaires, it was observed that all farms have farrow-to-finish production system, 40,5% have 10 to 20 years of production and 38,1% had between 100 and 500 sows. Serotyping these samples, it was noticed that S. suis is present in several Brazilian states, and the greater number of them belong to Minas Gerais, followed by São Paulo (10,4%) and Paraná (8,9%).The serotype 2 was the most frequent one (61,9%), followed by the serotypes 1, 3, 4, 7 and 8. The greater number of isolations occurred in brain (56,0%), followed by lung (9,8%) and septicemic cases (9,2%). Regarding to drugs’ sensitivity, the S. suis isolated samples were more sensitive to amoxicillin (96,4%), followed by lincomycin-spectinomycin (90,9%), ampicillin (87,2%), chloranphenicol (79,1%) and penicillin (71,0%).

Le but de cette étude était d'évaluer le potentiel antimicrobien de bactériocines pour le contrôle du pathogène porcin Streptococcus suis sérotype 2. Pour répondre au premier objectif de ce projet, la susceptibilité de S. suis à la nisine, une bactériocine produite par Lactococcus lactis, seule ainsi qu'en combinaison avec différents antibiotiques, a été démontré. Pour réaliser le second objectif, la purification et la caractérisation d'une bactériocine produite par une souche non-virulente de S. suis sérotype 2 ont été effectuées. La bactériocine a d'abord été purifiée par HPLC pour ensuite être caractérisée au niveau biochimique, ainsi que génique. Des études plus approfondies permettront d'évaluer la capacité de la nisine, de la suicine 90-1330 ou de leur souche productrice respective à prévenir des infections expérimentales à S. suis chez le porc en vue d'une potentielle application thérapeutique et préventive.

Streptococcus (S.) suis is one of the most important pathogens in pigs causing meningitis, arthritis, endocarditis and serositis. Furthermore, it is also an emerging zoonotic agent. In our previous work we identified a highly specific IgM protease in S. suis, designated IdeSsuis. The objective of this study was to characterize the function of IdeSsuis in the host-pathogen interaction. Edman-sequencing revealed that IdeSsuis cleaves the heavy chain of the IgM molecule between constant domain 2 and 3. As the C1q binding motif is located in the C3 domain, we hypothesized that IdeSsuis is involved in complement evasion. Complement-mediated hemolysis induced by porcine hyperimmune sera containing erythrocyte-specific IgM was abrogated by treatment of these sera with recombinant IdeSsuis. Furthermore, expression of IdeSsuis reduced IgM-triggered complement deposition on the bacterial surface. An infection experiment of prime-vaccinated growing piglets suggested attenuation in the virulence of the mutant 10ΔideSsuis. Bactericidal assays confirmed a positive effect of IdeSsuis expression on bacterial survival in porcine blood in the presence of high titers of specific IgM. In conclusion, this study demonstrates that IdeSsuis is a novel complement evasion factor, which is important for bacterial survival in porcine blood during the early adaptive (IgM-dominated) immune response.

Der zu den bedeutendsten Ektoparasiten des Schweines gehörende Erreger der Schweineräude, Sarcoptes scabiei var. suis, ist weltweit verbreitet und von wirtschaftlichen Interesse für Schweinezucht- und Mastbetriebe. Ziel dieser seroepidemiologischen Studie war die Gewinnung neuer Erkenntnisse über die Bestandsdynamik klinischer Räudeerscheinungen und dem Titerverlauf von IgG-Antikörpern gegen Sarcoptes scabiei var. suis im Serum von Sauen in Abhängigkeit vom Reproduktionszyklus (Trächtigkeit, Laktation, Besamung) und von Saugferkeln während der Säugeperiode sowie Aufzucht. Schlussfolgernd aufgrund der Ergebnisse dieser Studie eignen sich für die Kontrolle und Überwachung sowie Diagnostik der Sarcoptes-Räude von Beständen serologische Untersuchungen von Ferkeln bis zur zweiten Lebenswoche von räudeverdächtigen Sauen, „Jungsauen erster und zweiter Wurf“ sowie Sauen während der Trächtigkeit (vor allem letztes Drittel) und Deckeber. Eine Vorselektion auf räudeverdächtige Hautveränderungen sowie gesteigerte Kratzaktivitäten erhöht die Wahrscheinlichkeit serologisch positive Tiere zu identifizieren.

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.

O Actinobaculum suis é um dos principais micro-organismos relacionados a infecções de trato urinário em fêmeas suínas. As características de crescimento deste agente dificultam o isolamento bacteriano tradicional, o que pode tornar a sua prevalência subestimada. Este estudo teve por objetivos desenvolver a reação em cadeia pela polimerase (PCR) para detecção do A. suis, avaliar a sensibilidade e especificidade desta técnica e comparar seu desempenho com o isolamento bacteriano. Além disso, as cepas isoladas foram caracterizadas através do polimorfismo de comprimento de fragmentos amplificados (AFLP) e submetidas à determinação da concentração inibitória mínima para caracterização dos perfis de susceptibilidade antimicrobiana. Foram analisados 45 suabes prepuciais de machos e 192 urinas de fêmeas suínas provenientes de três granjas. Os resultados indicaram que a PCR desenvolvida foi específica para o A. suis e apresentou limiar de detecção entre 1,0 X 101 UF/mL e 1,0 X 102 UFC/mL. A frequência de A. suis encontrada através da PCR foi de 82,2% (37/45) nos suabes prepuciais e de 8,9% (17/192) nas urinas de fêmeas. No que se refere ao isolamento, nenhuma das amostras de urina foi positiva para o agente, enquanto 31,1% (14/45) dos suabes foram positivos. A partir das amostras positivas isoladas dos suabes prepuciais foram selecionadas 20 cepas de A. suis. Os perfis de susceptibilidade entre estas cepas foram semelhantes, no entanto diferiram dos isolados utilizados como controle e provenientes de uma fêmea com infecção urinária. A técnica de PCR foi mais eficiente que o isolamento na identificação de amostras positivas para A. suis. Através do AFLP com uma única enzima foi possível caracterizar todos os isolados e relacionar os dados obtidos com a origem das cepas e o perfil de resistência. Até o presente não há relatos na literatura de caracterização genotípica de A. suis através do AFLP ou detecção do agente através da PCR. / Actinobaculum suis is an important agent related to urinary infection in swine females. The growth characteristics of this agent hamper the traditional bacterial isolation, which can make their prevalence underestimated. The purpose of this study was to develop the polymerase chain reaction (PCR) for Actinobaculum suis detection, to evaluate the sensitivity and specificity of this technique and compare the results with bacterial isolation. Moreover, the isolates were characterized by amplified fragment length polymorphism (AFLP) and subjected to determination of minimum inhibitory concentration for characterization of the antimicrobial susceptibility profiles. Forty-five preputial swabs from boars and a hundred and ninety-two urine samples from sows of three herds were analyzed. The results indicate that the developed PCR was specific for A. suis, presenting a limit detection between 1.0 X 101 UFC/ml and 1.0 X 102 UFC/ml. A.suis frequency by PCR was 82.2% (37/45) in male preputial swabs and 8.9% (17/192) in females urine. Through traditional isolation, none of the urine samples were positive, while A.suis growing was observed in 31.1% (14/45) of the swabs. From the positive samples of the preputial swabs were selected 20 A.suis strains. The susceptibility profiles among these strains were similar, but differed from the female isolates used as control. The PCR technique was more effective than isolation for the A.suis detection. The AFLP with a single enzyme was able to characterize all isolates and relate the data obtained with the strains origin and resistance profile. Until present, there are no reports of genotypic characterization of A. suis strains through AFLP or agent detection by PCR.