Memoria para optar al Título Profesional de Médico Veterinario / El diagnóstico definitivo y específico de la brucelosis en caninos, causada por Brucella canis (B. canis), se realiza mediante el aislamiento de la bacteria desde muestras patológicas o fluidos. Sin embargo, como la enfermedad se caracteriza por presentar periodos abacterémicos, un cultivo negativo no excluye la posibilidad de infección. Debido a que se utiliza ampliamente la detección de anticuerpos frente a la infección y que tanto B. ovis y B. canis comparten componentes antigénicos con la cepa vacuna Brucella abortus RB51 (B. abortus CRB51), ésta podría ser usada como antígeno en el diagnóstico serológico de la enfermedad. En el presente estudio, se describe un enzimoinmunoensayo indirecto (ELISA-I) para el serodiagnóstico de brucelosis canina utilizando un extracto altamente purificado de lipopolisacárido rugoso (LPS-R) de Brucella abortus CRB51, cuyos resultados fueron comparados con la técnica contrainmunoelectroforesis (CIEF) que utiliza un antígeno extraído de una cepa rugosa de B. ovis, con diferentes componentes, incluyendo al LPS-R. En este estudio se incluyeron 156 sueros de perro, muestras para diagnóstico de brucelosis canina mediante CIEF, que fueron recibidas en el laboratorio de Microbiología de FAVET. El análisis de los resultados indicó asociación estadística entre ambas pruebas (p>0,05) para el diagnóstico de brucelosis canina. Además, ambas pruebas obtuvieron un alto índice de concordancia (K= 0,961). Estos resultados hacen del ELISA-I propuesto una prueba promisoria al utilizar un antígeno más purificado y entregar resultados cuantitativos

Memoria para optar al Título Profesional de Médico Veterinario / En el presente trabajo se describe la evolución de anticuerpos caninos contra Brucella canis, de dos perras inoculadas experimentalmente, mediante un ensayo inmunoenzimático indirecto (ELISA-I) usando como antígenos el lipopolisacárido rugoso (LPS-R) y proteínas citosólicas de Brucella abortus Cepa RB51. El objetivo fue discriminar el momento en que se hacen detectables los anticuerpos para cada ELISA-I y describir la evolución de éstos en el tiempo. Se utilizaron sueros de dos perras infectadas con B. canis, A y B, con un total de 20 y 16 muestras de sueros seriados, respectivamente. Para ambos ELISA-I se usaron líneas de corte obtenidas por metodología “Receiver-Operator Characteristic” (ROC), que determinaron la positividad o negatividad a la prueba. Ambos ELISA-I fueron capaces de detectar anticuerpos tempranamente y fue posible constatar su permanencia en el tiempo. La detección de anticuerpos mediante ELISA-I, tanto con antígeno de proteínas citosólicas como con antígeno LPS-R de B. abortus Cepa RB51, se mantuvo detectable, al menos, hasta la semana 38 post infección

Memoria para optar al Título Profesional de Médico Veterinario / La brucelosis es una enfermedad infectocontagiosa zoonótica que afecta a los mamíferos, con amplia distribución mundial y de gran importancia económica. Muchos países han implementado programas de control y/o erradicación dirigidos a especies domésticas. Sin embargo, Brucella spp. es capaz de infectar a animales silvestres y en Chile casi no existen registros tanto en aquellos de vida libre como en cautiverio. En zoológicos, es importante conocer los riesgos de las principales enfermedades, especialmente las zoonóticas, para dimensionar a lo que se enfrentan tanto quienes trabajan con los animales como quienes los visitan. Se realizó un estudio serológico en 80 ungulados en cautiverio, pertenecientes a 9 especies, para detectar anticuerpos contra Brucella spp. mediante las técnicas Rosa de Bengala y ELISA de Competencia. Ambas pruebas fueron similares en sus resultados. Para la técnica Rosa de Bengala todas las muestras fueron negativas, y para el ELISA de Competencia, 2 muestras reaccionaron positivamente con valores levemente superiores a la línea de corte validada para el bovino

La connaissance de la structure tridimensionnelle (3D) des protéines est une information capitale. Néanmoins, le nombre de protéines dont la structure 3D a été déterminée expérimentalement est cent fois plus faible que le nombre de protéines connues aujourd'hui. Cet écart ne pourra pas être comblé, car les techniques expérimentales de détermination de structure (diffraction de rayons X et résonance magnétique nucléaire) sont coûteuses et lentes (un an de travail en moyenne pour une seule protéine). Un moyen d'obtenir plus rapidement la structure 3D de protéines est de la prédire par des moyens bioinformatiques. La technique de prédiction la plus précise actuellement est la modélisation par homologie. Celle-ci est basée sur la similitude de structure entre deux protéines de séquences similaires. L'étape critique de cette méthode est l'étape d'alignement entre la séquence à modéliser et une séquence similaire de structure connue. Notre travail a consisté tout d'abord en la conception d'une nouvelle méthode d'alignement pairé très fiable. Cette méthode a ensuite été incluse dans un système automatique de modélisation par homologie: la bonne qualité des structures prédites par le système trouve en partie son origine dans le programme d'alignement utilisé. Enfin, nous avons appliqué notre système de modélisation automatique à la modélisation de toutes les protéines déduites du génome d'une bactérie pathogène étudiée dans notre unité de recherche: Brucella melitensis. Cela nous a conduit à créer une banque de données structurales et fonctionnelles consacrée au génome de cette bactérie. Cette banque de données est devenue un outil de travail indispensable pour plusieurs équipes de recherche européennes qui étudient Brucella melitensis.

Catalase-peroxidase (KatG) of Mycobacterium tuberculosis is a bifunctional heme enzyme that has been shown to play an important role in the activation of a first line drug, isoniazid (INH), used in the treatment of tuberculosis infection. Mutations in the katG gene have been found to be associated with INH resistance. The most commonly encountered mutation is the Ser315Thr point mutation. In this dissertation, the x-ray crystallographic structures of MtbKatG and the mutant enzyme KatG[S315T] are presented to explore the molecular basis of the INH activation and resistance. The structure is dimeric and contains a heme cofactor in each subunit of the dimer. The most important change in KatG[S315T] is due to the presence of the methyl group of the threonine 315 side chain, which is located at the narrowest part of the substrate channel. The protruding methyl group effectively constricts the accessibility to the heme by closing down the dimensions of the channel, constraining the substrate entrance. VirB11 of Brucella abortus is a hexameric ATPase that belongs to the type IV secretion system. The crystal structure of BaVirB11 was found to contain six molecules per asymmetric unit. The Walker A (P loop), His box, and Glu box from the C-terminal domain are located at the interface of the N- and C-terminal domain. A large conformational change was found in the linker region when compared with that of HP0525 structure, the VirB11 analogous from H. pylori. To elucidate the functional role of each domain, seven functional mutations were generated and used for biochemical studies. The GER motif and the linker region were found to be crucial for ATP hydrolysis activity of BaVirB11. Mutations in the GER motif (R101Q) and the linker region (R120E) of BaVirB11 completely abolish the ATP hydrolysis activity of the enzyme. The binding affinities of the two mutants to the ATP; however, are similar to that of the wild-type enzyme, indicating that mutation in the GER motif or the linker region has no effect on ATP binding.

Feral hogs (Sus scrofa) are considered an exotic, free-ranging ungulate distributed within numerous countries and continents to include the United States. The reproductive efficiency, lack of predators, land use practices for domestic livestock (e.g., feeding stations, introduced water sources, intense cropping practices, etc.), and diet are leading factors in the expansion of feral hogs throughout their range. Feral hogs negatively impact floral and faunal communities, agricultural lands, and residential and recreational areas to include concerns with public safety and disease transmission. My study objectives were to (1) assess feral hog abatement strategies by (A) evaluating trap designs with the inclusion of electrical fencing, and (B) evaluating candidate baits for feral hog-specificity, and (2) assess prevalence levels for feral hog diseases. I evaluated 3 corral trap designs differing in the addition of electric fence configurations. Feral hog capture success data were collected and used to determine trap design efficacy. Treatments evaluated included (A) control corral trap with no electrical configurations, (B) corral trap with 1 electrical leg, and (C) corral trap with 2 electrical legs. ANOVA analyses suggest no differences (df = 2, P = 0.758) between trap designs; however, length of trapping effort (i.e., the number of days that trapping occurred) was a significant (df = 6, P < 0.001) factor in determining trap success. Pre-baiting was an important factor in observed trapping success. Trapping success declined after fourth day of continuous trapping. I recommend short, intensive trapping efforts (e.g., <4 days) when using corral traps in feral hog abatement programs. I also evaluated 14 candidate baits (with and without repellant) replicated 40 times to determine feral hog specificity. Three evaluated baits (i.e., PIGOUT™ strawberry, corn, and rice) were selected (df = 2, P < 0.05) more frequently by feral hogs than other combinations. Non-target species (e.g., raccoons) visited baits with repellants less (df = 2, P < 0.05) than baits without repellants. Repellant had no direct impact on feral hog visitation at bait sites. Trapping data also suggests that grains commonly farmed in local or regional areas are more likely to be consumed by feral hogs and, therefore considered in baiting options. Finally, of 412 feral hogs captured, 86 were sampled for prevalence of pseudorabies and Brucella suis. The prevalence of pseudorabies and B. suis was 20.9% and 13.9%, respectively within the study area. Based on disease study results, I recommend that natural resource managers take necessary precautions to protect themselves by wearing protective equipment and equipment and properly cooking feral hog meat. Additionally, resource managers should properly administer vaccinations to domestic and companion animals, and restricting domestic and companion animals from areas of high risk (e.g., carcasses of dead hogs and wallows).

B rucella lipopolysaccharide is an important component of virulence in brucellosis. Recent research in macrophage models has shown that Brucella LPS does not behave like classical LPS by stimulating potent inflammatory responses. The central hypothesis of this work is that O-antigen is dynamic signaling molecular and participates in complex interactions with the host to promote productive infection. A corollary to this is that the host environment is dynamic, and Brucella has evolved mechanisms to cope with changing environments. In an effort to understand the contribution of Brucella LPS to virulence and pathogenesis, the function of a metabolic locus important in the synthesis of LPS has been demonstrated and complemented. The spontaneous loss of LPS expression has been characterized. Contribution of LPS to acquisition of the host environment in tissue culture and mouse models has been explored. This work demonstrated that genes outside the O-antigen biosynthesis ( manBA) cluster contribute to LPS biosynthesis. Further high frequency mutation involving manBA is partly responsible for observed dissociation of Brucella strains. Finally, work herein attempts to look at the role of LPS in acquisition of the host environment and shows that LPS is important for recruiting particular cell populations within a host model of brucellosis.

Despite being amongst the most common zoonotic diseases in the world, brucellosis is a neglected disease for which an approved vaccine for human use does not exist. Thus far, the traditional approaches to Brucella antigen selection for subunit vaccine development have yielded unacceptable results. In this work, we evaluated the predictive ability of a multistep Brucella antigen selection process with in vitro immunological and invasion assays and in vivo protection experiments. Initial in silico screening for antigens was performed via genomic sequence analysis where 27 Brucella melitensis open reading frames (ORF) coding for outer membrane proteins bearing MHC epitopes, adhesin and conserved properties were identified. Evidence for a role in any aspect of Brucella virulence (i.e., invasion, co-regulation/expression with known Brucella virulence factors, intracellular adaptation) was then used to narrow the list of candidate antigens. To further increase confidence in the candidate ORF putative role in Brucella pathogenesis, differential expression of candidate ORF was evaluated using previously generated global transcriptomics data in in vitro HeLa and in vivo bovine models of acute Brucella infection. Protein expression in the E. coli heterologous system resulted in the successful expression of OmpW, BtuB, Omp22, Hia, and FlgK. With regards to virulence, the two proteins with the highest predicted adhesin scores conferred an invasive phenotype to the non-invasive BL-21 E. coli strain in alveolar epithelial cells. From an immunogenicity standpoint, all proteins elicited IgG production in Brucella-exposed goats, mouse and humans. Antigen-specific recall responses in splenocytes from C57BL/6 mice immunized with a cocktail of the three proteins with highest MHC scores revealed a mixed Th1/Th2 response with a comparatively greater Th1 response. In protection studies, subcutaneous (SQ) immunization with BtuB, Hia and FlgK, individually, promoted bacterial clearance following a robust intraperitoneal challenge dose of Brucella melitensis 16M. In addition, single SQ inoculation of FlgK enhanced protective efficacy of the vaccine strain B. abortus S19. In contrast, immunization of mice with the three protective antigens in a cocktail formulation elicited immune responses but no protection against intraperitoneal challenge with Brucella melitensis 16M in the spleen and liver. In conclusion, our results indicate that our combinatorial in silico, in vitro and in vivo antigen selection and identification modeling approach provides strong evidence for prediction of Brucella protective antigens, and represent a novel strategy with broad application to other major pathogens.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1949. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves [i]-viii).

Made available in DSpace on 2014-06-11T19:32:52Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-15Bitstream added on 2014-06-13T18:44:25Z : No. of bitstreams: 1 magajevski_fs_dr_jabo.pdf: 1942398 bytes, checksum: b212ff62ed36cb609ef85e31f2538b3b (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A leptospirose e a brucelose são duas das mais importantes enfermidades da esfera reprodutiva em bovinos, podendo ser transmitidas durante a utilização de técnicas de reprodução assistida. Este trabalho visa pesquisar a presença e a ação da Leptospira spp. e da Brucella spp. em fetos e oócitos bovinos. Foram colhidas amostras do sangue de 512 vacas encaminhadas para o abate em um frigorífico do Estado de São Paulo; dessas, 212 foram utilizadas para a pesquisa de Leptospira spp. e Brucella spp. nos fetos, e 300 foram utilizadas para pesquisa desses agentes no líquido folicular e nos oócitos. Amostras de órgãos fetais (rim, fígado, baço e pulmão), conteúdo estomacal fetal, placenta materna e líquido folicular (LF) foram submetidas às técnicas de cultivo em meio próprio para crescimento de Leptospira spp (Fletcher) e Brucella spp. (Brucella-ágar) e à técnica de reação em cadeia da polimerase multiplex (PCRm) para ambos os agentes. Os oócitos provenientes de vacas sorologicamente negativas para leptospirose e brucelose foram maturados em meio contaminado com Leptospira interrogans sorovar Pomona, sendo uma parte em meio sem antibiótico e outra com antibiótico. Os oOOtos provenientes de fêmeas sorologicamente positivas para leptospirose foram maturados em meio de maturação esterilizado, sendo uma parte sem antibiótico e outra com antibiótico, e oOOtos provenientes de vacas sororreagentes para brucelose foram maturados em meio estéril sem antibiótico. Após a maturação, parte dos oócitos foi encaminhada para PCRm, e outra parte fixada em lâmina e corada pelas técnicas de Levaditi e- imunoistoquímica para a pesquisa de Leptospira spp.. Entre as 512 vacas avaliadas, 234 (45,70%) foram reagentes na soroaglutinação microscópica (SAM) e 50 (9,76%) sororreagentes na prova do antígeno acidificado tamponado (AA T), sendo 34 (6,64%)... / Leptospirosis and brucellosis are two of the most important diseases of the reproductive sphere in bovines, being able to be transmitted during the use of assisted reproduction techniques. This work aims to research the presence and the action of Leptospira spp. and Brucella spp. in bovine fetus and oocytes. Blood samples of 512 slaughtered cows, were collected in a slaughterhouse in the State of São Paulo. Of these, 212 were used for the research of Leptospira spp. and Brucella spp. in the fetus, and 300 were used for research of these agents in the follicular liquid and the oocytes. Samples of fetal organs (kidney, liver, spleen and lung), fetal stomach content, maternal placenta and follicular liquid (FL) were submitted to the techniques of cultivation for the growth of Leptospira spp. (Fletcher) and Brucella spp. (Brucella-agar) and the technique of PCRmultiplex for both agents. The oocytes from serological negative cows for leptospirosis and brucellosis were matured in a contaminated media with Leptospira interrogans serovar Pomona, being a part without antibiotic and another one with antibiotic. The oocytes from serologically positive females for leptospirosis were matured in a sterilized environment, being a part without antibiotic and another with antibiotic and oocytes from cows seroreacting, from brucellosis were matured in sterile environmenta without antibiotic. Afier the maturation, part of the oocytes were directed to PCRm and another part settled in the blade and dyed by the Levaditi technique and immunohistochemical for the research of Leptospira spp.. Among the 512 evaluated cows, 234 (45,70%) reacted to the microscopic seroagglutination (MAT) and 50 (9,76%) reacted to the Rose Bengal Plate Test (RBPT), being 34 (6,64%)...(Complete abstract, access undermentioned eletronic address)