No universo da pecuária leiteira, a mastite representa um importante desafio, sendo responsável por perdas econômicas consideráveis relacionadas principalmente com redução na produção de leite. O gênero Staphylococcus assume elevada importância como agente etiológico das mastites devido à sua ampla distribuição e freqüência de ocorrência. Foram coletadas amostras de leite de fêmeas bovinas com mastite subclínica para exames microbiológicos e contagem de células somáticas (CCS). Após o isolamento e identificação dos micro-organismos, as amostras positivas foram submetidas a análises das medianas das CCS, testes de susceptibilidade \"in vitro\" frente a diferentes antimicrobianos, assim como pesquisa de genes codificadores das adesinas - genes que codificam para proteína ligadora de colágeno (cna), proteína ligadora de laminina (eno), proteína ligadora de elastina (ebp), proteína ligadora de fibrinogênio (fib), proteína A ligadora de fibronectina (fnbA), proteína B ligadora de fibronectina (fnbB) e proteína associada à formação de biofilme (bap), por meio da Reação em Cadeia da Polimerase (PCR). De acordo com os achados do presente estudo, dentre as bactérias do gênero Staphylococcus isoladas, os S. aureus foram verificados com maior freqüência, seguido por Staphylococcus coagulase-negativos - S. intermedius, S. chromogens e S. warneri. Com respeito às medianas das CCS, o gênero Streptococcus spp. apresentou o maior valor. O perfil de sensibilidade e resistência aos antimicrobianos testados foi semelhante entre as espécies de Staphylococcus coagulase-positivos e negativos, sendo os antimicrobianos cefalexina, cefalotina e ceftiofur os que apresentaram maior frequência de sensibilidade, e penicilina, amoxicilina e ampicilina os que apresentaram maior resistência. Com exceção do (fnbA), todos os outros fatores de virulência estudados foram detectados, sendo os genes eno, fib e a associação dos genes \"eno/fib/bap\" os mais freqüentemente detectados. Nas amostras coletadas dos tanques de refrigeração não foram detectadas todas as espécies de Staphylococcus spp. isoladas dos quartos mamários. Tais informações acerca do assunto permitem o desenvolvimento de estratégias mais eficientes de tratamento e controle desta enfermidade, possibilitando o aumento da produtividade leiteira. / In the world of dairy cattle, mastitis is a major challenge, accounting for economic losses related mainly to reduction in milk production. The genus Staphylococcus assumes greater importance as a etiologic agent of mastitis due to its wide distribution and frequency of occurrence. Milk samples were collected from cows with subclinical mastitis to microbiological examinations and somatic cell count (SCC). After isolation and identification of microorganisms, positive samples were analyzed for the medians of SCC, tested for susceptibility \"in vitro\" against different antimicrobials and polymerase chain reaction will be used to search for genes encoding adhesins - genes that code for collagen-binding protein (cna), lamininbinding protein (eno), elastin-binding protein (ebp), fibrinogen-binding protein (fib), fibronectin-binding protein A (fnbA), fibronectin-binding protein B (fnbB) and protein associated with biofilm formation (bap). According to the findings of this study, among the bacteria of the genus Staphylococcus isolated, S. aureus were found most frequently, followed by coagulase-negative Staphylococcus - S. intermedius, S. chromogens and S. warneri. With respect to the medians of CCS, the genus Streptococcus spp. showed the highest. The profile of sensitivity and resistance to antimicrobials was similar among species of coagulase-positive and negative Staphylococcus, and antimicrobial cephalexin, cephalothin, and ceftiofur showed higher frequency sensitivity. Penicillin, amoxicillin and ampicillin showed the highest resistance. With the exception of (fnbA), all other virulence factors were detected, with genes eno, fib and the association of genes \"eno/fib/bap\" the most frequently detected. The samples collected from the cooling tanks were not detected all species of Staphylococcus spp. that were isolated from the mammary glands. Such information on the subject allow the development of more efficient strategies for treatment and control of this disease, allowing for increased milk production.

Ram lamb epididymitis, a serious reproductive disease of sheep, is caused principally by bacteria belonging to the genera Haemophilus and Actinobacillus. Six bacteria were studied: the American Type Culture Collection (ATCC) of Actinobacillus seminis (ATCC 15768), ATCC of Actinobacillus actinomycetemcomitans (ATCC 29522), field isolates of A seminis (86722 and 4101) and field isolates of Actin obacillus- like bacteria (Y136 and D107). The objectives of this study were to quantitate the adhesion of these 6 bacteria to bovine kidney epithelial cells (BKECs) and ram epididymal epithelial cells (REECs), evaluate the effect of rabbit polyclonal antibody prepared against ATCC 15768 (PoAb 15768) on bacterial adherence to BKECs and REECs, and partially characterize the adhesins present on these bacteria. In a bacterial adhesion assay (BAA), strain and species differences were noted. The number of bacteria adhering to each BKEC ranged (rom a low of 4.27 ± 1.00 (Actinobacillus-like 0107) to a high of 31.84 ± 2.00 (A seminis 86722). The number of bacteria adhering to each REEC ranged from a low of 3.05 ± 0.34 (Actinobacill us-like 0107) to a high of 21.61 ± 2.03 (Actinobacillus like Yl36). In a bacterial inhibition assay (BIA), PoAb 15768 inhibited the adhesion of ATCC 15768 to both BKECs and REECs by 5%. This same antiserum inhibited the adhesion of ATCC 29522 to BKECs by 14.5% and to REECs by 22%. The inhibition of A seminis 86722 adherence to BKECs and to REECs was less than 14% and 35%, respectively. For A seminis 4101, Actinobacillus-like Y136, and Actinobacillus-like 0107, PoAb 15768 failed to prevent adhesion to either BKECs or REECs. When the 6 bacteria were analyzed by autoradiography, 2 (Actinobacillus-like 0107) to 8 (ATCC 29522) potential adhesins were identified. However, the pathogenicity has not been firmly established for many Actinobacillus species and Actinobacillus-like bacteria. The potential adhesins identified in this study were not unequivocally confirmed as bacterial adhesins. An in vit ro model may facilitate the recognition of potential adhesins used by Actinobacillus species and Actinobacillus-like bacteria and may eventually lead to the development of an efficacious bacterin to prevent epididymitis in ram lambs at risk.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 120-131). Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 96-101).

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Weiping Zhang / Enterotoxigenic Escherichia coli (ETEC) strains are the most common bacterial cause of diarrhea. ETEC bacterial adherence to the small intestinal epithelial cells and delivery of enterotoxins cause diarrhea in children living in developing countries and international travelers. Currently, there are no vaccines licensed for ETEC associated children’s diarrhea and travelers’ diarrhea. Recently, toxoid fusion 3xSTa[subscript N12S]-mnLT[subscript R192G/L211A] (toxoid fusion), adhesin MEFA (multiepitope fusion antigen) CFA/I/II/IV (CFA MEFA), and toxoid-adhesin MEFA CFA/I/II/IV-3xSTa[subscript N12S]-mnLT[subscript R192G/L211A] (CFA-toxoid MEFA) are demonstrated to induce neutralizing antitoxin and/or anti-adhesin antibodies in intraperitoneal (IP) or subcutaneous (SC) immunized mice, suggesting these antigens are potential candidates for ETEC subunit vaccines. However, these antigens have not been examined for immunogenicity using intradermal (ID) or intramuscular (IM) routes, the routes perhaps are more suitable for human vaccine administration. In this study, toxoid fusion 3xST[subscript aN12S]-mnLT[subscript R192G/L211A], CFA/I/II/IV MEFA, alone or combined, or toxoid-adhesin MEFA CFA-3xSTa[subscript N12S]-mnLT[subscript R192G/L211A] were ID or IM immunized to mice (8 mice per group) induced antigen-specific antibodies were titrated, and antibody neutralization activities were assessed in vitro. Data showed that mice ID or IM immunized with the toxoid fusion 3xSTa[subscript N12S]-mnLT[subscript R192G/L211A] antigen developed anti-LT and anti-STa antibodies and mice immunized with the CFA/I/II/IV MEFA developed antibody responses to all seven adhesins (CFA/I, CS1-CS6). In addition, mice co-administered ID or IM with toxoid fusion 3xSTa[subscript N12S]-mnLT[subscript R192G/L211A] and CFA/I/II/IV MEFA, or with toxoid-adhesin MEFA CFA-3xSTa[subscript N12S]-mnLT[subscript R192G/L211A] developed antibodies to both toxins and all seven adhesins. Antibody neutralization studies of the serum samples of the immunized mice showed that the induced antibodies neutralized enterotoxicity of LT and STa and/or inhibited adherence of ETEC or E. coli bacteria producing any of these seven adhesins. These data confirmed immunogenicity of these ETEC subunit vaccine target antigens and provide useful information for vaccine development against ETEC diarrhea.

Neisseria meningitidis (N. meningitidis) is a Gram-negative commensal bacterium colonizing nasopharynx of about 10 % of healthy individuals, which can cause invasive diseases, such sepsis and meningitis, upon occasional penetration into bloodstream. Pathogenesis of N. meningitidis appears to be directly related to conditions of limited iron availability. Under these conditions two proteins of unknown function: FrpC and FrpD, are synthesized. FrpD is a highly conserved lipoprotein of N. meningitidis anchored to the bacterial outer membrane. It is known that FrpD tightly binds the FrpC protein, which belongs to the Repeat-in-Toxin (RTX) protein family and may act as bacterial exotoxin. However, the mechanism of FrpD-FrpC interaction and the exact function of this complex are unknown due to the absence of structural information on these proteins. Therefore, we set out to determine the structure of FrpD and provide insights into its interaction mechanism with FrpC and structure-functional relationships of these two proteins. We determined the first crystal and solution structures of the FrpD protein. We found that atomic structures of FrpD reveal a novel protein fold. We uncovered the structure-function relationships underlying the mechanism of interaction between the FrpD and FrpC proteins and tested the putative function of the FrpD-FrpC1-414 complex in vitro. Finally, we proposed the putative function of the FrpD-FrpC1-414 complex as a new minor adhesin of N. meningitidis, which mediates the bacterial adhesion to the host epithelial cells and facilitate the colonization. Our work constitutes the first step in clarifying the molecular basis of the FrpD-FrpC interaction and sets the base for further investigation of the role of FrpD and FrpC in the virulence mechanism of N. meningitidis.

Neste trabalho foi estuda a interação da E. coli patogênica aviária (APEC), agente etiológico da colibacilose aviária, e células não fagocitárias. A APEC é uma ExPEC (E. coli extra-intestinais), grupo que também inclui a UPEC (E. coli uropatogênica) e a NMEC (E. coli de meningite neonatal). Foi analisado o comportamento de 8 cepas APEC - MT78, IMT2470, A2363, UEL31, UEL13, UEL17, IMT5155, UEL29 - frente a duas linhagens de células não-fagocitárias, fibroblastos aviários CEC-32 e células endoteliais humanas EAhy926. Foi realizada a genotipagem de 33 genes associados à virulência, verificou-se capacidade de associação (adesão e invasão), de invasão e de multiplicação intracelular, de citotoxicidade e de ativação das caspases 3/7 das cepas após infecção de fibroblastos aviários. Foi observado que enquanto todas as cepas foram capazes de aderir aos fibroblastos aviários, somente a cepa MT78 foi capaz de invadí-los em níveis comparáveis à bactéria invasiva Salmonella Typhymurium SL1344. As cepas APEC não induziram ativação de capases 3/7, nem foram citotóxicas aos fibroblastos. Uma vez que a cepa invasiva, MT78, e a não invasiva, IMT2470, apresentam genótipos de virulência muito similares, foi realizado o estudo da expressão por RT-PCR dos genes de virulência da bactéria crescida com e sem fibroblastos aviários, por 3 h. Os resultados mostraram a expressão de adesinas, sideróforos e protectinas/estruturas de resistência ao soro para ambas as cepas na ausência e presença de fibroblastos. A análise da expressão dos genes fimH, ompA, ibeA e gimB pela técnica RT-qPCR revelou a repressão do gene fimH na MT78, mas indução do mesmo na IMT2470. Já as invasinas, gimB e ibeA, estavam induzidas em MT78 e reprimidas em IMT2470, enquanto o gene ompA estava sendo expresso em ambas as cepas. A expressão das invasinas gimB e ibeA explica em parte o fenótipo invasivo da MT78. No presente estudo também foi analisado o comportamento das 8 cepas APEC em relação ao perfil de associação, invasão e multiplicação intracelular ao infectarem células EAhy926. As cepas não mostraram capacidade de invadir as células mas apresentaram um nível de associação muito superior ao dos fibroblastos aviários (até 14 vezes superior para algumas cepas). Este trabalho mostrou que o ensaio in vitro com CEC-32 é um modelo adequado para o estudo da interação celular entre APEC e células eucarióticas e agregou mais conhecimento sobre o patotipo. / In this work the interaction between avian pathogenic E. coli (APEC), the etiological agent of avian colibacillosis, and non-phagocytic cells was studied. APEC is an ExPEC (extraintestinal E. coli), a group that also includes UPEC (uropathogenic E. coli) and NMEC (E. coli neonatal meningitis). We analysed the behavior of 8 APEC strains - MT78, IMT2470, A2363, UEL31, UEL13, UEL17, IMT5155, UEL29 - against two non-phagocytic cell lines, avian fibroblasts (CEC-32) and human endothelial cells (Eahy926). Strains were genotyped for 33 virulence associated genes, and investigated the association capacity (adhesion and invasion), the invasion ability, intracellular multiplication, cytotoxicity and activation of caspases 3/7 of the strains after infecting avian fibroblasts. While all strains were able to adhere to avian fibroblasts, only the strain MT78 was able to invade them at levels comparable to the invasive bacterium Salmonella Typhymurium SL1344. APEC strains could not induce activation of caspases 3/7, nor were cytotoxic to fibroblasts. Since the invasive MT78 and the non-invasive IMT2470 strains presented very similar virulence genotypes, the expression of virulence genes of the bacteria grown in the absence and presence of avian fibroblasts by 3 h was analysed by RT-PCR. Results showed the expression of adhesins, siderophores and protectins/serum resistance structures for both strains in the two culture conditions, with and without fibroblasts. Analysis of the expression of fimH, ompA, ibeA and gimB by RT-qPCR revealed the repression of fimH in MT78, but the induction in IMT2470. In relation to ibeA and gimB invasins, they were induced in MT78 but repressed in IMT2470, while the ompA gene was expressed in both strains. The expression of invasins partly explains the invasive phenotype of MT78. It was also analysed the behaviour of the strains in relation to the association profile, invasion and intracellular multiplication when infecting EAhy926 human endothelial cells. The strains showed no ability to invade endothelial cells but showed a high level of association (up to 14 times higher than for fibroblasts cells for some strains). This study showed that the CEC-32 in vitro model is suitable for the study of cellular interaction between APEC and eukaryotic cells, and added more knowledge about the pathotype.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Sporothrix schenckii é um fungo dimórfico e agente etiológico da esporotricose, uma micose profunda que apresenta diferentes manifestações clínicas. As diversas manifestações clínicas desta e de outras doenças infecciosas podem ser relacionadas ao status imune do hospedeiro, a fatores de virulência do patógeno ou a diferentes genótipos. Dados anteriores do nosso grupo demonstram que diferenças na expressão de adesinas do S. schenckii para fibronectina estão diretamente relacionadas à virulência de diferentes cepas. Neste trabalho visamos avaliar caracteres morfológicos bioquímicos e genotípicos de doze isolados de geofílicos, zoofílicos e antropofílicos de S. schenckii, de diferentes origens geográficas, que apresentam diferentes graus de virulência. Foi analisada a morfologia das formas de micélio e levedura de cada isolado. Foi observado que a fase de micélio dos isolados estudados apresentaram morfologia típica com hifas finas e septadas, com conídios obovóides ou ovóides alongados. As leveduras apresentaram pleomorfismo típico da espécie, com células variando do formato ovóide ao alongado. Verificamos ainda a expressão de adesinas para fibronectina e laminina, do antígeno gp70 e, o padrão de bandas antigênicas reconhecidas por anticorpos IgG presentes em soro de pacientes com esporotricose ou de camundongos infectados. Para isso, foram extraídas proteínas de superfície da forma de levedura de cada isolada, sendo os extratos ensaiados por Western blot. Nestes ensaios observamos que os isolados mais virulentos de S. schenckii expressavam mais adesinas para fibronectina e laminina. A presença da gp70 foi detectada em dez dos doze isolados, sendo que apenas os isolados zoofílicos não expressam esta glicoproteína. O padrão antigênico foi variável entre os isolados, não havendo clara relação com a origem e/ou distribuição geográfica. Os dados fenotípicos foram confrontados com dados genotípicos. Para isso, sequenciamos os loci da calmodulina (CAL) e do Internal Transcribed Spacer 1/2 (ITS 1/2) a fim de averiguar se haviam diferenças genotípicas entre os isolados estudados. As análises do sequenciamento do loci CAL e ITS, contudo, apontam a divisão dos isolados em duas espécies filogenéticas, S. schenckii e S. brasiliensis não correlacionada com a distribuição geográfica dos mesmos. Nosso estudo reforça a hipótese de haver uma correlação entre virulência e expressão de adesinas, porém, sem qualquer relação entre a distribuição geográfica dos isolados zoofilicos, antropofílicos ou geofílicos, bem como dos genótipos encontrados. / The dimorphic fungus Sporothrix schenckii is the etiological agent of sporotrichosis, a deep mycosis that presents different clinical manifestations. The diverse manifestations of this and other infections can be related the host immunity, virulence factors or different genotypes of the pathogen. Previous data of our group demonstrated that differences in the expression of adhesins to fibronectin are directly related to the virulence of the different strains of S. schenckii. In the present work we have evaluated the morphological, biochemical and genotypic characteristics of twelve strains of S. schenckii (isolated from human and cat cases of sporotrichosis and from environment) from different geographic regions that present distinct virulence levels. The mycelium and yeast phases of each strain were morphologically analyzed. The strains has presented the typical morphology, with thin and septated hyphae. The conidia exhibited obovoidal or ovoidal elongated form. The yeast phase presented the ovoid or elongated cell form. Furthermore, we have verified the adhesins expression to fibronectin, to laminin, to gp70 antigen and to the antigenic bands pattern of all protein extracts, recognized by patients or infected mice sera antibodies. For this, the proteins were extracted from the surface of each strain yeast phase and assayed by Western blot technique. We have observed that the most virulent strains of S. schenckii expressed more adhesins to fibronectin and to laminina than less virulent strains. The presence of the gp70 was confirmed in ten isolates of twelve. Just strains isolated from infected cats did not present this glycoprotein. The antigenic bands pattern was variable between the different extracts, with no clear correlation with origin or geographical distribution of the isolates of S. schenckii. In order to cross phenotypic and genotypic data we have sequenced the calmodulin loci (CAL) and the Internal Transcribed Spacer 1/2 (ITS 1/2) in order to check if there was differences between the strains studied. In this analysis we found that this strains are divided in two species, S. schenckii and S. brasiliensis, again with no correlation with the geographical distribution. Our study reinforces the hypothesis on the correlation between adhesins expression pattern and virulence levels with no connection among geographical distribution or genotype of the different strains of S. schenckii.