A mortalidade fetal é uma causa importante de perdas reprodutivas em animais domésticos e tem impacto significativo na rentabilidade de um sistema de produção animal. No Brasil, poucas informações são disponíveis sobre as principais causa de aborto em bovinos. O presente estudo resultou em cinco artigos. O primeiro artigo descreve as principais causas de aborto bovino diagnosticadas no Setor de Patologia Veterinária da Universidade Federal do Rio Grande do Sul no período 2003-2011. Neste período um total de 490 fetos bovinos foi analisado. Causas específicas de aborto foram encontradas em 46,7 % dos casos. Infecções por protozoários, em especial Neospora caninum acometeram 33 % dos casos (162/490). Bactérias em 6,3 % (31/490), seguidas por fungos 0,8% (4/490) foram causas adicionais de abortos. Em dois fetos (0,4 %), coinfecções por dois agentes foram identificadas. Causas não infecciosas foram observadas em 3 % dos abortos e malformações congênitas a 2,6%. No segundo artigo estima-se a ocorrência de abortos por Brucella abortus em bovinos na região sul, caracterizando os achados macroscópicos, histopatológicos, bacteriológicos, imuno-histoquímicos e de PCR desta enfermidade. No terceiro artigo relata-se a infecção por Geotrichum candidum em feto bovino abortado associado a lesões de pele e pulmonares. A ocorrência de malformações congênitas múltiplas em um feto bovino abortado é relatada no quarto artigo e casos de condrodisplasia tipo Dexter em fetos bovinos abortados são descritos no quinto. / The fetal mortality is an important cause of reproductive failure in domestic animals and has a significant impact on the profitability of a livestock production system. In Brazil, little information is available on the main cause of abortion in cattle. This study resulted in five articles. The first article describes the main causes of abortion in cattle diagnosed in the period January 2003 to december 2011. A total of 490 fetuses, from several Brazilian states, were evaluated. Specific causes of abortion were found in 46,7 % of cases. Protozoan abortions especially Neospora caninum were detected in 33 % (162/490) of the cases. Bacterial abortions corresponded to 6,3 % (31/490), followed by fungal 0,8 % (4/490). In two aborted fetuses (0,4 %), a co-infection with two agents could be identified. Noninfectious diseases could be associated with 3 % of the abortions and congenital malformations with 2,6%. In the second article estimated the incidence of abortion by Brucella abortus in cattle in the southern region, characterizing the macroscopic findings, histological, bacteriological, immunohistochemical and molecular aspects of this disease. The third article relates a Geotrichum candidum infection in aborted bovine fetus associated with skin and lung lesions. The occurrence of multiple congenital malformations in an aborted bovine fetus is reported in the fourth article and cases of Dexter chondrodysplasia type of aborted fetuses are described in the fifth.

Submitted by Mario BC (mario@bc.ufrpe.br) on 2018-02-22T14:28:38Z No. of bitstreams: 1 Adriana Soares Leite.pdf: 443532 bytes, checksum: aecae8c9b89ee037b16d8d03ce6267a9 (MD5) / Made available in DSpace on 2018-02-22T14:28:38Z (GMT). No. of bitstreams: 1 Adriana Soares Leite.pdf: 443532 bytes, checksum: aecae8c9b89ee037b16d8d03ce6267a9 (MD5) Previous issue date: 2004-06-09 / An epidemiological study was performed to evaluate the prevalence of serum antibody to B. abortus, BVDV, BHV – 1 and tuberculin sensitivity to M. bovis, and the association among these infections. Sera from 241 dairy buffaloes were analyzed by buffered plate antigen test (BPAT), serum agglutination test (SAT) and by 2-mercaptoethanol (2-ME) for the diagnosis of Brucella abortus, virusneutralization (VN) for BVDV and BHV – 1 and comparative cervical tuberculin test for M. bovis. The results showed 32,34% of positive samples to B. abortus, 24,07% of positive tuberculin reactors, 76,76% of reactive samples to BVDV and 49,79% to BHV – 1. Association was found between reagent and non reagent animals to the tests to B. abortus and BHV – 1 (p<0,001). / Foi realizado um estudo epidemiológico para determinar a prevalência de anticorpos séricos anti-Brucella abortus e a associação com as infecções por Mycobacterium bovis, vírus da diarréia viral bovina (BVDV) e vírus da rinotraqueíte infecciosa bovina - IBR (BHV–1). Foram analisadas 241 amostras séricas de bubalinos com aptidão leiteira no Município de Taipu – RN, empregando o teste do antígeno acidificado tamponado (AAT) e as provas de soroaglutinação lenta em tubos (SAL) e 2–mercaptoetanol (2-ME) para o diagnóstico da brucelose; prova de virusneutralização (VN) para detecção de anticorpos para BVD e IBR. Os animais foram também submetidos ao teste cervical comparativo para tuberculose. Os resultados mostraram 32,34% de amostras soropositivas para B. abortus, 24,07% de animais com reação positiva ao teste alérgico para Tuberculose, 76,76% de animais sororreagentes para BVDV e 49,79% para BHV – 1. Foi detectada associação entre animais reagentes e não reagentes aos testes para B. abortus e BHV – 1 (p<0,001).

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-05-10T14:39:12Z No. of bitstreams: 1 andrealmeidasantosduch.pdf: 1562353 bytes, checksum: c681c7dd0bb3bc45cdb7bc06579fadb5 (MD5) / Rejected by Adriana Oliveira (adriana.oliveira@ufjf.edu.br), reason: Falta resumo em português on 2016-06-15T19:03:46Z (GMT) / Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-06-16T11:27:16Z No. of bitstreams: 1 andrealmeidasantosduch.pdf: 1562353 bytes, checksum: c681c7dd0bb3bc45cdb7bc06579fadb5 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-07-13T14:12:36Z (GMT) No. of bitstreams: 1 andrealmeidasantosduch.pdf: 1562353 bytes, checksum: c681c7dd0bb3bc45cdb7bc06579fadb5 (MD5) / Made available in DSpace on 2016-07-13T14:12:36Z (GMT). No. of bitstreams: 1 andrealmeidasantosduch.pdf: 1562353 bytes, checksum: c681c7dd0bb3bc45cdb7bc06579fadb5 (MD5) Previous issue date: 2015-05-29 / O objetivo desse trabalho foi estimar a prevalência de Brucella spp. em propriedades produtoras de Queijo Minas Artesanal da microrregião do Serro. Avaliou-se a presença do patógeno (presença de DNA) em queijos com períodos de maturação entre 4 e 8 dias, em temperatura ambiente, em queijarias cadastradas pelo Instituto Mineiro de Agropecuária (IMA), que possuem assim o aval do órgão para o comércio estadual. Os resultados das análises de Reação de Cadeia da Polimerase (PCR) mostraram que a prevalência da Brucella spp. em propriedades produtoras de Queijos Minas Artesanal do Serro foi de 30,9% (17/55). Os produtos de DNA amplificados por PCR, de amostras de queijos positivas na PCR foram sequenciados, para diferenciação entre bactérias de origem do campo ou vacinais. O sequenciamento demonstrou 100% de homologia com amostras do campo de Brucella spp. Todas as amostras de queijos foram provenientes de rebanhos testados e controlados por meio de exames sorológicos anuais para o monitoramento de Brucella spp., segundo determinação da legislação vigente, e apresentaram atestados de controles negativos de veterinários autônomos habilitados. A presença de DNA de Brucella spp. nos queijos sugere uma suposta deficiência no atual programa de controle do patógeno nesses rebanhos e que medidas de identificação e controle mais rigorosas deveriam ser adotadas como medida de segurança alimentar tanto em nível de rebanho, como na matéria prima e no produto acabado. / The main objective of the study is to estimate Brucella spp. prevalence in dairy herds involved in artisanal cheeses production in Serro’s micro-region, Minas Gerais state, named of “Queijo Minas Artesanal” (QMA). The pathogen's evidences (presence of DNA) in cheeses indexed by the “Instituto Mineiro de Agropecuária – IMA” with ripening periods at normal temperature between 4 and 8 days was evaluated. The results of the polymerase chain reaction (PCR) showed that the DNA of Brucella spp. prevalences in Serro's QMA was of 30.9% (17/55). The products of amplified DNA were sequenced for differentiating the bacteria’s source: if it came from the environment or from the vaccine. The DNA sequencing demonstrated 100% of homology with environment samples of Brucella ssp. All sample of cheeses were from cattletested and controlled through annual serological tests for the monitoring of brucelosis and presented negative control´s certificates delivered by autonomous veterinaries. The presence of DNA Brucella spp. in analyzed cheeses suggests a supposed deficiency of brazilian brucelosis control's program in monitoring this specific herds and that stronger measures of control should be adopted in the herds, ingredients and also in the final product as a food safety measure.

Memoria para optar al Título Profesional de Médico Veterinario / En el presente estudio se describe el desarrollo y validación de un Ensayo inmunoenzimático indirecto (ELISA-I) para el diagnóstico serológico de la infección por Brucella ovis (B. ovis) en ovinos, utilizando proteínas citosólicas de Brucella abortus RB51 (B. abortus RB51) como antígeno. Se probaron dos tipos de placas de poliestireno, NUNC 69620 y NUNC Maxisorp, dos tampones de sensibilización de diferente pH con dos alternativas de conjugado, el anticuerpo monoclonal anti IgG-caprino/ovino (Sigma A9452) y la proteína G (Sigma P81170). Las concentraciones óptimas de antígeno citosólico, sueros y conjugados fueron determinadas mediante titulación del tipo checkerboard. Para la validación del ensayo se utilizaron 55 sueros de ovinos considerados como infectados/expuestos, positivos a examen clínico, a un ELISA-I comercial, a la fijación del complemento (FC) y a la prueba de reacción en cadena de la polimerasa (PCR) y 338 sueros de ovinos no infectados, provenientes de un rebaño ovino libre de infección, negativos a la prueba de FC. El punto de corte diagnóstico fue determinado a través del método de las características del tipo receptor-operador (ROC). El ELISA-I desarrollado con anticuerpo monoclonal como conjugado mostró mejores valores de sensibilidad (90,9%) y especificidad (95,9%). Estos resultados, a pesar de ser significativos, son deficientes para ser considerados de utilidad diagnóstica y lograr un adecuado control de la infección en rebaños ovinos. La baja sensibilidad obtenida al utilizar proteínas citosólicas como antígeno estaría relacionada con la escasa exposición al antígeno y la corta memoria inmunológica que generarían este tipo de proteínas en un animal infectado.

Brucellosis is a bacterial infection that primarily affects livestock and can also be transmitted to humans. In the Greater Yellowstone Ecosystem (GYE), elk (Cervus canadensis) and bison (Bison bison) are habitual carriers of Brucella abortus, which arrived to the region with cattle over a century ago. The disease was eliminated from cattle in the United States through widespread control efforts, but is now periodically transmitted back to cattle on open rangelands where they can come into contact with fetal tissues and fluids from disease-induced abortions that occur among elk during the late winter and spring. In Wyoming, south of Yellowstone National Park, there are 23 supplemental feedgrounds that operate annually and feed the majority of the region’s elk during a portion of the winter. The feedgrounds are controversial because of their association with brucellosis and may be shuttered in the future in part due to the arrival of chronic wasting disease. Using data collected at these feedgrounds, this study investigates the role of winter feedgrounds in the ecology of this host-pathogen relationship: it evaluates the full reproductive costs of the disease to affected elk, how herd demography influences pathogen transmission, and assesses management strategies aimed at reducing pathogen spread among elk. Using blood tests for pregnancy status and brucellosis exposure in female elk, I demonstrated a previously undocumented fertility cost associated with the pathogen which is not due to abortions, but which nearly doubles the estimated fertility cost to affected individuals. I also built mechanistic transmission models using time-series disease and count data from feedgrounds. Within that framework, I assessed various management actions including test-and-slaughter of test-positive elk, which I found to be counterproductive due to rapid recovery times and the protective effects of herd immunity. The overall picture that emerges of winter feedgrounds is one of imperfect practicality driven by social and political consideration, not pathogen control. These results illustrate the underappreciated importance that recruitment and population turnover have on the transmission dynamics of brucellosis in elk, a pathogen which itself flourishes in the reproductive tracts of individual animals and thus impacts vital rates at the population level. Together, this study contributes to the field of disease ecology using a unique long term disease data set of free-ranging wild ungulates.

The present investigation was made in the Laboratories of microbiology of the Ability of Cattle Sciences of the Polytechnic Superior School of Chimborazo, with samples obtained in the Municipal Camal Riobamba and the Community of Tunshi San Nicolás. The incidence of Brucellosis was analyzed in six more important cattle areas of the county using the antigens Brucella abortus and Rosa of Flare. The experimental units were obtained under a systematic sampling. The experiment had a duration of 4 months. The experimental results underwent the following analyses: variance, percentages, stockings, standard deviation, limit for the estimate error. According to carried out analysis of the obtained results, you determines that the analysis technique by means of antigen Brucella abortus, detected more cases of brucellosis (16.59% ± 4.61%) regarding the technique with Rosa of Flare (9.98% ± 3.59%). The biggest percentage of suspicious cases met with the technical Rosa of Flare (9.57% ± 3.35%) regarding the technique of antigen Brucella abortus (5% ±2.5%). According to the sex we have that in more quantity they presented the females with 9.16% in relation to the males with 7.1%, this with the antigen Brucella abortus. With Rosa of Flare we have for the females 5.42% and the males 4.42%. We carry out a study of 17 animals of the Community of Tunshi San Nicolás, we find completely free of this bacterial infection, with the two studied methods.

Brucella are Gram-negative intracellular bacteria that cause abortion and infertility in livestock and chronic disease in humans. The Centers for Disease Control and Prevention (CDC) categorizes them as class B pathogens due to their zoonotic potential. Currently, there are no efficacious Brucella vaccines for humans available. Very few studies have focused on identifying protective vaccines against respiratory exposure. Protection by B. abortus rough vaccine strains RB51 and RB51SOD is through strong CD4⁺ Th₁ and CD8⁺ Tc₁ adaptive immunity. However, limited information is available on how they stimulate innate immunity. This knowledge is critical for improving these vaccines for their potential use in humans. Dendritic cells (DCs) play a crucial role bridging innate and adaptive immunity. Therefore, enhancing the ability of rough vaccine strains to induce DC maturation and function could be critical for upregulating protective T-cell responses. Herein, we demonstrated that live vaccine strain RB51 induced significantly better (p≤0.05) DC maturation and function in vitro and upon intranasal inoculation in vivo compared to strain RB51SOD or strain 2308. Due to safety concerns of live vaccines, irradiated and heat killed vaccines were also tested; only live strain RB51 infected DCs induced significant (p≤0.05) DC function based on TNF-α and IL-12 secretion. DC activation occurs through Toll-like receptors (TLRs) 2, 4 and 9. Our study reported that strain RB51 induced significant (p≤0.05) DC activation compared to strain 2308, which was not dependent on a specific TLR. However, strain RB51 induced TNF – α production was TLR2 and TLR9 dependent and IL-12 production was TLR2 and TLR4 dependent. TLR4 KO mice had significantly (p≤0.05) higher number of strain RB51 colonies present at day 14 post infection. By unraveling the innate immune responses to Brucella, the ultimate goal of these studies is to develop a protective vaccine for animals and people against respiratory challenge. As such, we tested several vaccination strategies. Despite enhanced DC activation and function achieved by vaccine strains, they failed to protect mice against intranasal challenge with strain 2308. Future experiments will address host-pathogen interaction at the lung microenvironment and elucidate immune mechanisms that will enhance protection against aerosol exposure. / Ph. D.

Livestock across the world contract several infectious diseases of both bacterial and viral origin. Swine brucellosis caused by Brucella suis and swine influenza caused by Influenza A virus affect both domestic and feral swine populations. Both the diseases have zoonotic potential to cause disease in humans with serious complications apart from inflicting huge economic losses. Infected feral swine can also act as a source of spread and outbreak where the disease is not endemic. At present, there is no vaccine available for swine brucellosis. The currently used swine influenza vaccine may not be effective against influenza strains like the recent H1N1 strain that caused a pandemic. To develop an effective bivalent vaccine for swine against these two diseases, a leucine auxotroph of the USDA approved vaccine B. abortus strain RB51 was constructed along with leuB gene complementing plasmid pNS4 to over-express antigens from Brucella and influenza. This antibiotic resistance free system over-expressed Brucella derived antigens SOD, L7/L12 and WboA in three different constructs. Against a virulent challenge of B. suis, the candidate vaccine strain over-expressing both SOD and WboA protected mice more significantly than the control group and was also found to be better protective than other candidate vaccine strains over-expressing either SOD and L7/L12 together or SOD alone. Immunoassays (ELISA) suggested that the protection afforded is Th1 type mediated immune response, as cytokine IFN-γ and IgG2a antibody sub-isotype was observed in the splenocyte culture supernatant and serum samples respectively. The strain RB51leuB platform was not expressing influenza derived antigens Hemagglutinin (HA) and Nucleoprotein (NP) when screened for expression by immunoblot. Influenza antigens, HA, NP and ectodomain of matrix protein M2e, were not found to be expressing even after optimizing their codon usage to suit Brucella tRNA preference. However, RT-PCR showed that the influenza genes mRNA were produced. In conclusion, this dissertation describes the construction of an environmentally safe antigen over-expression platform and successful employment of the system as a candidate vaccine in protecting mice against B. suis challenge. This new platform is a potential candidate for developing vaccines against other infectious diseases of livestock. This document also discusses alternate strategies for expressing influenza antigens in a Brucella platform. / Ph. D.

Brucella abortus is amongst the top 5 zoonotic diseases worldwide. The overall goal of this research is to generate a safe and effective vaccine for humans. Brucella abortus strain RB51, approved for use in cattle, provides protection by initiating a strong T-helper 1 (Th1) type response is a candidate vaccine. Based on a model for aerosol exposure mice were vaccinated intranasally (IN) with strain RB51 and challenged IN with B. abortus strain 2308, strain RB51 did not protect. Protection against Brucella is mediated through TLRs 2, 4 and 9. The addition of TLR 2 or TLR 4 and a trend with TLR9 agonists with intranasal RB51 vaccination significantly increased bacterial clearance in the lung after strain 2308 challenge. Therefore, we hypothesized that combining TLR agonists 2, 4, and 9 with strain RB51 IN would upregulate protection and clearance in the lung against strain 2308 challenge (IN), by upregulating the DC1 and CD4 Th1 and CD8 immune response. This study showed that protection is not upregulated by combining all TLR agonists. Overall the addition of TLR 2 and 4 vs. TLR 2, 4 and 9 agonists affects the immune response and impacts the level of clearance. Our data support the development of a DC1 Th1 CD8 response, based on serology, and both DC and T-cell activation and function by the group which received the TLR 2 and 4 agonists and to a lesser degree the group receiving TLR 2, 4, and 9 agonists. Additional studies are warranted to further define the differential mechanisms and endpoints of protection. / Master of Science

<i>Brucella abortus</i> is capable of resisting the microbicidal mechanisms of phagocytic cells and growing within phagocytic cells, usually macrophages. <I>B. abortus</i>, like several other intracellular bacteria responds to the hostile environment in macrophages by producing heat shock proteins (HSPs) which are induced by environmental stresses. Bacterial HSPs are very immunogenic, eliciting both cellular and humoral immune responses in the infected host. The significance of host cellular and protective immune responses directed against these proteins is currently unresolved. Baculovirus recombinants were generated in <i>Sf9</i> insect cells for <i>B. abortus</i> HSPs and the protein expression was optimized. Humoral (Western blot), cell mediated (CMI, IFN-g- release by splenocytes, and CD3+CD4+, CD3+CD8+ T cell/ total splenocytes ratios) and protective immune responses of BALB/c mice (challenge with virulent <i>B. abortus</i> 2308) against these recombinants, against <i>B. abortus</i> superoxide dismutase (SOD) and ribosomal L7/L12 proteins, inoculated alone or in various combinations with complete Freund's, Ribi and recombinant IL-12 as adjuvants, were analyzed. Vaccinia virus-GroEL recombinant as priming immunogen, followed by baculovirus-GroEL-Ribi booster, was explored. Androstenediol, an immune up-regulator, was tested for its ability to induce resistance against challenge. None of the mice inoculated with individual, divalent or trivalent HSP-expressing <i>Sf9</i> cells combined with Freund's were protected against challenge and the <i>Sf9</i> cell-induced response masked the recombinant protein-specific CMI responses. Recombinant HSPs were purified and combined with Ribi. Although significant IFN-g release was induced by immunization with the HtrA-Ribi combination, no mice were protected against challenge. Priming with vaccinia virus-GroEl recombinant and boosting with purified baculovirus-GroEL protein-Ribi combination did not induce protection. Androstenediol did not enhance in vivo resistance to challenge. IL-12 alone did not activate splenocytes but induced significant IFN-g release in mice when combined with killed <i>B. abortu</i>s RB51 vaccine, purified recombinant HtrA or purified SOD proteins, or L7/L12 expressing <i>Escherichia coli</i> cells. Significant protection was induced by SOD combined with IL-12. No correlation was seen between IFN-g release by splenocytes and protection against challenge in the SOD/IL-12-immunized mice. The results suggest that <i>B. abortus</i> HSPs are not highly immunogenic in mice and though various immune responses may be induced by one or another HSPs, protective immune response, unfortunately, is not among them. The results of this study reflect the difficulties in experimenting with immune responses against single or a limited number of recombinant <i>B. abortus</i> proteins. This is particularly true when the task includes induction of a protective immune response and finding significant correlation between different types of immune response assays. / Ph. D.