Biological and molecular characteristics of the pathogenic mollicute, Spiroplasma penaei, isolated from the hemolymph of infected Pacific white shrimp, Penaeus vannamei, were investigated. The doubling times of a S. penaei were 6.13 h (2% NaCl) and 3.43 h (no salt) under aerobic conditions, and 6.63 h (2% NaCl) and 3.22 h (no salt) under anaerobic conditions. Small diffuse white colonies with granular centers, surrounded by small satellite colonies that appeared embedded in the agar matrix, were detected on solid M1D medium (2% Noble agar) under aerobic conditions at 28°C. The genome size of the S. penaei was 1778 Kb, as determined by pulsed-field gel electrophoresis using undigested DNA. Reduction of virulence of S. penaei was not detected in serial passage 24 and 76 isolates but passage 131 isolate was attenuated as indicated by the number of surviving shrimp and histological findings of challenged P. vannamei. Toxicity was not detected in supernatant fractions of M1D medium cultures of S. penaei isolates. The most predominant host responses to the S. penaei reference isolate and to serial passage isolates were hemocytic nodules and hemocytic infiltration observed in hematoxylin and eosin-stained histological sections. Transmission electron microscopy of the lymphoid organ of experimentally infected P. vannamei depicted S. penaei without cell wall and free in the cytoplasm of lymphoid organ cells. The lesions observed in histological sections were verified by in situ hybridization using a digoxigenin (DIG)-labeled probe specific to the spiralin gene of Spiroplasma spp. Evolutionary relationship trees, based on five partial DNA sequences of 16S rDNA, 23S rDNA, 5S rDNA, gyrB, rpoB genes and two complete DNA sequences of 16S-23S and 23S-5S ISR, were constructed using the distance-based Neighboring-Joining method with Kimura-2-parameter substitution model. The NJ trees based on all DNA sequences investigated in this study positioned S. penaei in the Citri-Poulsonii clade and corroborates the observations by other investigators using the 16S gene. Pairwise genetic distance calculation between sequences of spiroplasmas showed S. penaei to be closely related to S. insolitum and distantly related to Spiroplasma sp. SHRIMP from China.

O fungo leveduriforme Malassezia pachydermatis está presente na microbiota da pele dos animais. Alterações na imunidade da pele do hospedeiro fazem com que a levedura sofra adaptações modificando a expressão de fatores de virulência, causando otite, dermatite e até mesmo infecção sistêmica em animais e humanos. O tratamento de M. pachydermatis é relativamente simples, à base de antifúngicos azólicos. No entanto, o aparecimento de cepas resistentes e de infecções recorrentes tem gerado preocupação recentemente. Drosophila melanogaster tem sido um modelo promissor no estudo de microrganismos, apresentando duas vias de sinalização da resposta imune, a via Imd contra bactérias gram-negativas e a via Toll contra bactérias gram-positivas e fungos, as quais desencadeiam a produção dos peptídeos antimicrobianos locais e sistêmicos. A virulência de M. pachydermatis em moscas D. melanogaster wild-type e Toll-deficientes foi testada com inóculos nas concentrações entre 103 e 107 unidades formadoras de colônia (UFC)/ml e a infecção ocorreu pela punção com agulha no tórax das moscas, que foram colocadas em frascos com alimento e incubados à 29 °C por sete dias, com contagem diária da sobrevivência. Ainda foram realizados a contagem de UFC/ml/mosca e a histopatologia após os sete dias de observação. As moscas wild-type se mostraram resistentes à infecção. Curvas de mortalidade de moscas Toll-deficientes, em concentrações a partir de 104 leveduras/ml, diferiram estatisticamente do grupo controle. Após o sétimo dia de infecção, moscas wild-type inoculadas com 1 x 107 UFC/ml tinham carga fúngica variando entre 0,33 a 1 x 102 UFC/mosca, enquanto em moscas Toll-deficientes a carga fúngica variou de 2,3 x 103 a 1,3 x 104 UFC/mosca. Portanto, D. melanogaster Toll-deficientes são suscetíveis à infecção por M. pachydermatis devido à falta do sistema imune para o reconhecimento e montagem da resposta imune frente ao fungo, semelhante ao que ocorre em pacientes imunocomprometidos, dado o sistema imune conservado entre os mamíferos e a mosca-das-frutas. Deste modo, D. melanogaster é um modelo promissor e inovador para estudos de patogenicidade e de prospecção de fármacos frente a M. pachydermatis. / The yeast Malassezia pachydermatis is present in the normal microbiota of the skin of animals. Changes in the host's immunity cause the yeast to undergo adaptations modifying the expression of virulence factors, causing otitis, dermatitis and even systemic infection in animals and humans. The treatment of M. pachydermatis is relatively simple, based on the use of azole antifungals. However, the emergence of resistant strains and recurrent infections has generated concern recently. Drosophila melanogaster has been a promising model in the study of microorganisms, presenting two pathways of immune response signaling, the Imd pathway against gram-negative bacteria and the Toll pathway against gram-positive bacteria and fungi, which trigger the production of local and systemic antimicrobial peptides. The virulence of M. pachydermatis in wild-type and Toll-deficient D. melanogaster flies was tested with inoculum concentrations ranging between 103 and 107 colony forming units (CFU)/ml and the infection occurred by needle puncture in the thorax of the flies, which were placed in vials with food and incubated at 29 °C for seven days, with daily survival counts. Fungal burden counts and the histopathology were also performed after seven days of observation. Wild-type flies were resistant to infection. Mortality curves of Toll-deficient flies, at concentrations superior of 104 yeasts/ml, differed statistically from the control group. After the seventh day of infection, wild-type flies inoculated with 1 x 107 CFU/ml had fungal load ranging from 0.33 to 1 x 102 CFU/fly, while in Toll-deficient flies the fungal load ranged from 2.3 x 103 to 1.3 x 104 CFU/fly. We concluded that Toll-deficient D. melanogaster flies are susceptible to infection by M. pachydermatis due to lack of immune system for the recognition and assembly of immune response to the fungus, similar to that occurring in immunocompromised patients, given the conserved immune system among mammals and fruit flies. Therefore, D. melanogaster is a promising and innovative model for pathogenicity studies and prospection of drugs against M. pachydermatis.

Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen worldwide. Chlamydia spp. infect epithelial cells of the respiratory, intestinal, and reproductive tracts. Chlamydial infections in women may lead to pelvic inflammatory disease, ectopic pregnancy, chronic pelvic pain, and infertility. In addition to infecting infection the female reproductive tract (FRT), Chlamydia also infects the gastrointestinal tract (GIT) of animals and humans. In mice Chlamydia muridarum disseminates from the FRT to the GIT via internal routes and in a stepwise manner. Initially Chlamydia spreads from the FRT to infect the FRT-draining iliac lymph nodes (ILNs), then the spleen, and then the GIT. The first step of this dissemination (FRT to ILN) is mediated by tissue CD11c+ DCs. Chlamydia transport from ILN to the spleen is dependent on cell transport and is mediated by sphingosine 1-phosphate (S1P) signaling. The third step of Chlamydia transport from the spleen to the GIT is significantly hindered in splenectomized mice. However, which cells mediate this transportation of the second and the third step remain unknown. Using mouse-specific C. muridarum as a model pathogen we show that following depletion of CD8+ T cells or monocytes, Chlamydia dissemination to the spleen and the GIT is significantly hindered. Furthermore, this study reveals that Chlamydia may infect various cell types which then mediate its dissemination internally. It remains to be determined what role systemic dissemination may have in Chlamydia pathogenesis.

Submitted by Lucas Bocchini Rodrigues Alves null (lucasbocchini@gmail.com) on 2017-12-05T19:45:59Z No. of bitstreams: 1 Dissertação_Lucas_Bocchini_Rodrigues_Alves.pdf: 48834271 bytes, checksum: 5031c0180284664e9a7c99d9701586b4 (MD5) / Submitted by Lucas Bocchini Rodrigues Alves null (lucasbocchini@gmail.com) on 2017-12-11T18:47:10Z No. of bitstreams: 1 Dissertação_Lucas_Bocchini_Rodrigues_Alves.pdf: 48834271 bytes, checksum: 5031c0180284664e9a7c99d9701586b4 (MD5) / Approved for entry into archive by Karina Gimenes Fernandes null (karinagi@fcav.unesp.br) on 2017-12-13T11:36:05Z (GMT) No. of bitstreams: 1 rodriguesalves_lb_jabo.pdf: 48291587 bytes, checksum: dba569cb90d71bd85abca6cc394cb228 (MD5) / Made available in DSpace on 2017-12-13T11:36:05Z (GMT). No. of bitstreams: 1 rodriguesalves_lb_jabo.pdf: 48291587 bytes, checksum: dba569cb90d71bd85abca6cc394cb228 (MD5) Previous issue date: 2017-07-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / RESUMO – Salmonella Gallinarum (SG) é um patógeno hospedeiro-específico que causa o tifo aviário, doença sistêmica severa que é considerada uma das principais preocupações da indústria avícola mundial. Quando infecta a ave, SG utiliza mecanismos de evasão para sobreviver e replicar no interior de macrófagos. Nesse contexto, os genes phoPQ codificam o sistema regulatório de dois componentes (PhoPQ) que regula genes de virulência responsáveis pela adaptação de Salmonella spp. a fatores antimicrobianos como baixo pH, peptídeos antimicrobianos e baixas concentrações de cátions bivalentes. No presente estudo, objetivou-se investigar a função desses genes para SG. Assim, uma estirpe de SG com genes phoPQ defectivos (SG ∆phoPQ) foi construída e sua patogenicidade avaliada em aves poedeiras de 20 dias de vida susceptíveis ao tifo aviário. SG ∆phoPQ não causou sinais clínicos nem mortalidade em aves desafiadas oralmente, sendo não-patogênica. Ademais, essa estirpe não foi recuperada de fígados e baços. Por outro lado, aves desafiadas subcutaneamente com a estirpe mutante tiveram alterações patológicas discretas a moderadas e baixas contagens bacterianas em tecidos de fígado e baço. A partir dos dados, observa-se que SG ∆phoPQ é atenuado para aves o que sugere que ambos os genes são importantes durante a infecção sistêmica em aves por SG. / ABSTRACT – Salmonella Gallinarum (SG) is a host-restrict pathogen that causes fowl typhoid, a severe systemic disease that is one of the major concerns to the poultry industry worldwide. When infecting the bird, SG makes use of evasion mechanisms to survive and to replicate within macrophages. In this context, phoPQ genes encode a two-component regulatory system (PhoPQ) that regulates virulence genes responsible for adaptation of Salmonella spp. to antimicrobial factors such as low pH, antimicrobial peptides and deprivation of bivalent cations. Herein, we aimed to investigate the role of the mentioned genes to SG. Thus, a phoPQ-depleted SG strain (SG ∆phoPQ) was constructed and its virulence assessed in twenty-day-old laying hens susceptible to fowl typhoid. SG ∆phoPQ did cause neither clinical signs nor mortality in birds orally challenged, being non-pathogenic. Furthermore, this strain was not recovered from livers or spleens. On the other hand, chickens challenged subcutaneously with the mutant strain had discreet to moderate pathological changes and also low bacterial counts in liver and spleen tissues. These findings show that SG ∆phoPQ is attenuated to susceptible chickens and suggest that both genes are important during chicken systemic infection by SG.

En production avicole, S. Senftenberg connue pour être souvent associée au couvoir, est devenue très fréquente dans les élevages de volaille. L’objectif de la thèse est donc de comprendre les causes de cette persistance. Les résultats présentés suggèrent que l’existence de souches persistantes au sein du sérovar Senftenberg peut être à l’origine de l’augmentation de sa prévalence dans les élevages. Nous avons en effet identifié des souches présentant des phénotypes distincts en termes de persistance dans les caeca de poulets. Les souches persistantes ont la capacité d’induire un portage intestinal similaire à S. Enteritidis.Toutefois, les résultats in vivo ont montré qu’à la différence de S. Enteritidis, les souches de S.Senftenberg sont incapables d’induire une forte infection systémique chez le poulet et la souris,probablement due à leur faible capacité à résister aux cellules immunitaires. La comparaison entre les souches persistantes et non-persistantes n’a montré aucune différence de survie dans le contenu et le mucus caecal. Cependant, les souches persistantes ont une meilleure capacité à coloniser et persister dans les tissus d’animaux ce qui pourrait être une explication possible à l’augmentation de leur persistance. Ce caractère pourrait donc présenter un risque pour la santé humaine étant donné que ces bactéries peuvent être présentes chez les animaux avant l'abattage et la transformation des aliments. / In poultry production, S. Senftenberg was associated to the hatchery, but has recently become morefrequent in poultry farms. The aim of our study is to explain the increased persistence of this serovar. Our findings suggest that the existence of persistent strains within the serovar Senftenberg could explain its recent emergence in poultry flocks. We identified strains showing different persistence phenotypes inchicken caeca. The persistent strains are able to induce an intestinal asymptomatic carrier state in chicks similar to S. Enteritidis. However, the in vivo analysis showed that S. Senftenberg strains, contrary to S.Enteritidis are unable to induce a strong systemic infection in infected mice and chickens which could be in part related to their low capacity to resist to immune cells. The comparison between persistent and nonpersistents trains showed no difference in their ability to grow in the caecal content and mucus. However,persistent strains are more able to colonize and persist in chickens and mice tissues, which could be a possible explanation for their increased persistence. This trait might thus present a human health risk as these bacteria could be present in animals before slaughter and during food processing.

Opportunistic bacterial pathogens are present in the intestines of all mammals. These bacteria are symbionts to a certain extent, but under certain conditions these organisms can be deadly. Intestinal opportunistic pathogens encompass many genera and include organisms such as those in the Bacteroides fragilis group (i.e. B. fragilis and B. thetaiotaomicron), Escherichia coli, and Clostridium perfringens, resulting in an array of diseases and serious health risks. Typically these diseases affect individuals in poor or weakened health (elderly, immuno-compromised, neonates, etc.) but can affect healthy individuals as well. The intestinal tract is the main area of infection for these bacteria, however some of these organisms can be involved in wound infections, septicemia, urinary tract infections, and meningitis. This study focused on three areas: 1) Analysis of differences in gene expression between Bacteroides and Escherichia coli, in order to learn more about promoter structure, 2) Establishment of a diabetic mouse model for use in examining bacterial synergy during a polymicrobial infection, and 3) Characterization of Escherichia coli 360A and evaluation of the role of several virulence factors and environmental modulators in the pathogenesis of this strain. We used a newly developed lux gene reporter to evaluate gene expression in Bacteroides. We observed that there are barriers in both transcription and translation initiation that appear to limit the expression of foreign genes in Bacteroides. We were able to establish a mouse model for studying synergy during a polymicrobial infection and observed that E. coli 360A provided synergy towards B. fragilis NCTC 9343. These experiments also showed that the longer a mouse is afflicted with the complications of diabetes the more susceptible it is to polymicrobial infections. Systemic infections were used to evaluate the contribution of several virulence factors and environmental modulators in the pathogenesis of E. coli 360A. The results showed that a strain lacking both virulence factors CNF1 and HlyA, the terminal oxidase cytochrome o, or a double cyo/cyd mutant were, deficient in survival in the spleen, but not the liver of BALB/c mice. / Ph. D.

No description available.

No description available.

Le genre bactérien Salmonella regroupe plus de 2500 sérovars, mais peu sont responsables de pathologies humaines. Salmonella enterica sérovar Typhi (S. Typhi) est reconnu pour son importance médicale à travers le globe. S. Typhi cause la fièvre typhoïde chez l’Homme, une maladie infectieuse létale caractérisée par la dissémination systémique de la bactérie vers des organes du système réticulo-endothélial. La fièvre typhoïde représente un fardeau pour la santé mondiale, notamment auprès des pays en développement où les conditions sanitaires sont désuètes. La situation se complique davantage par l’apparition de souches résistantes aux antibiotiques. De plus, les deux vaccins licenciés sont d’efficacité modérée, présentent certaines contraintes techniques et ne sont pas appropriés pour les jeunes enfants et nourrissons. La phase systémique de l’infection par Salmonella repose sur sa survie dans les macrophages du système immunitaire. Dans ce compartiment intracellulaire, la bactérie module les défenses antimicrobiennes grâce à de multiples facteurs de virulence encodés dans son génome. Les mécanismes moléculaires sollicités sont complexes et finement régulés. Malgré les progrès scientifiques réalisés précédemment, plusieurs incompréhensions persistent au sujet de l’adaptation de ce pathogène dans les macrophages de l’hôte. Pour mieux concevoir les déterminants génétiques de S. Typhi impliqués dans l’interaction avec ces cellules, une stratégie de sélection négative a été appliquée afin de vérifier systématiquement l’effet direct des gènes pendant l’infection. En premier temps, une librairie de mutants par transposon chez S. Typhi a été créée pour l’infection de macrophages humains en culture. Après 24 heures d’infection, la présence des mutants fut évaluée simultanément par analyse sur des biopuces de Salmonella. Au total, 130 gènes ont été sélectionnés pour leur contribution potentielle auprès des macrophages infectés. Ces gènes comptaient des composantes d’enveloppe bactérienne, des éléments fimbriaires, des portions du flagelle, des régulateurs, des facteurs de pathogenèse et plusieurs protéines sans fonction connue. En deuxième temps, cette collection de gènes a dirigé la création de 28 mutants de délétion définie chez S. Typhi. Les capacités d’entrée et de réplication intracellulaire de ces mutants au sein des macrophages humains ont été caractérisées. D’abord, les macrophages ont été co-infectés avec les mutants en présence de la souche sauvage, pour vérifier la compétitivité de chacun d’eux envers cette dernière. Ensuite, les mutants ont été inoculés individuellement chez les macrophages et leur infectivité fut mesurée comparativement à celle de la souche sauvage. Sommairement, 26 mutants ont présenté des défauts lorsqu’en compétition, tandis que 14 mutants se sont montrés défectueux lorsque testés seuls. Par ailleurs, 12 mutants ont exposé une déficience lors de l’infection mixte et individuelle, incluant les mutants acrA, exbDB, flhCD, fliC, gppA, mlc, pgtE, typA, waaQGP, STY1867-68, STY2346 et SPI-4. Notamment, 35 nouveaux phénotypes défectueux d’entrée ou de survie intracellulaire chez Salmonella ont été révélés par cette étude. Les données générées ici offrent plusieurs nouvelles pistes pour élucider comment S. Typhi manipule sa niche intracellulaire, menant à l’infection systémique. Les gènes décrits représentent des cibles potentielles pour atténuer la bactérie chez l’humain et pourraient contribuer au développement de meilleures souches vaccinales pour immuniser contre la fièvre typhoïde. / The bacterial genus Salmonella holds over 2500 serovars, but few are responsible for human pathologies. Salmonella enterica serovar Typhi (S. Typhi) is recognized across the globe for its medical importance. S. Typhi causes typhoid fever in humans, a lethal infectious disease characterized by systemic dissemination of the bacteria to organs of the reticulo-endothelial system. Typhoid fever represents a burden for public health, notably in developing countries where sanitary conditions are obsolete. The situation is further complicated by the appearance of strains resistant to antibiotics. Moreover, both of the licensed vaccines are of moderate efficiency, present certain technical constraints and are not appropriate for young children and newborns. The systemic phase of infection by Salmonella relies on its survival within macrophages of the immune system. In this intracellular compartment, the bacterium modulates antimicrobial defenses thanks to multiple virulence factors encoded within its genome. Molecular mechanisms taking place are complex and finely regulated. Despite scientific advances made previously, many misunderstandings persist concerning the adaptation of this pathogen within host macrophages. To better conceive the genetic determinants of S. Typhi involved in interaction with these cells, a negative selection strategy was applied to systematically verify the direct effect of genes during infection. Firstly, a library of transposon insertion mutants in S. Typhi was created for infection of cultured human macrophages. After 24 hours of infection, the presence of mutants was evaluated simultaneously by analysis on Salmonella microarrays. In total, 130 genes were selected for their potential contribution within infected macrophages. These genes included bacterial envelope components, fimbrial elements, portions of the flagellum, regulators, pathogenesis factors, and many proteins of unknown function. Secondly, this collection of genes led to the creation of 28 defined deletion mutants in S. Typhi. The ability of entry and intracellular replication of these mutants within human macrophages were characterized. To start, macrophages were coinfected with mutants in the presence of the wild-type strain, in order to verify the competitiveness of each of them against the latter. Then, mutants were inoculated individually into macrophages and their infectiveness was measured in comparison with the wild-type strain. In summary, 26 mutants presented defects when in competition, whereas 14 mutants were shown defective when tested alone. Furthermore, 12 mutants exposed a deficiency during mixed and individual infection experiments, including mutants acrA, exbDB, flhCD, fliC, gppA, mlc, pgtE, typA, waaQGP, STY1867-68, STY2346, and SPI-4. In particular, 35 new defective phenotypes of Salmonella entry or intracellular survival were revealed in this study. Data generated here provides significant novel insight for elucidating how S. Typhi manipulates its intracellular niche, leading to systemic infection. Genes described represent potential targets for attenuating the bacteria in the human host and could contribute to the development of better vaccine strains to immunize against typhoid fever.

No description available.