Identification et caractérisation de gènes chez Salmonella enterica sérovar Typhi impliqués dans l’interaction avec les macrophages humains.

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.

Production and immunogenicity of selected proteins of Salmonella Enteritidis

Au cours des dernières années, Salmonella Enteritidis est devenus les sérotypes les plus souvent isolés chez les patients canadiens, les cas étant liés à la consommation de viande de poulet et d’œufs crus. Les vaccins tués commercialement disponibles pour la volaille, stimulent mal l'immunité mucosale, tandis que l'utilisation de vaccins vivants reste controversée. Par conséquent, un vaccin sous-unitaire par voie orale peut être une solution. Cinq protéines bactériennes ont été choisies comme candidates potentielles et identifiées, soit Glyceraldehyde-3-phosphate dehydrogenase, Enolase, Lipoamide dehydrogenase, DNA protection during starvation protein et Elongation factor-Tu. Notre objectif a été de produire et de purifier ces protéines et de démontrer leur immunogénicité. Les gènes des protéines ont été amplifiés et clonés dans le vecteur pQE-30 pour expression dans Escherichia coli M15. La purification a été effectuée par FPLC. Des poules pondeuses SPF ont été séparées en 6 groupes et injectées par voie intramusculaire à different âges avec une des 5 protéines, ou le PBS chez le groupe témoin. Les œufs ont été ramassés pendant l'expérience et du sang a été prélevé à 36 semaines d'âge. Les anticorps IgY ont été extraits à partir du jaune d'oeuf et du sérum, et les IgA à partir du blanc d'oeuf. Des immunodots, westernblots et ELISA ont évalué l'immunogénicité des protéines et les niveaux d'anticorps induits . Nous avons constaté que ces cinq protéines pourraient stimuler la production d'anticorps spécifiques in vivo. GAPDH, Enolase et DPS ont induit des titres d'anticorps plus élevés que LpdA et EF-Tu.

Formulation of a live bacterial vaccine for stable room temperature storage results in loss of acid, bile and bile salt resistance

Live bacterial vaccines have great promise both as vaccines against enteric pathogens and as heterologous antigen vectors against diverse diseases. Ideally, room temperature stable dry formulations of live bacterial vaccines will allow oral vaccination without cold-chain storage or injections. Attenuated Salmonella can cross the intestinal wall and deliver replicating antigen plus innate immune activation signals directly to the intestinal immune tissues, however the ingested bacteria must survive firstly gastric acid and secondly the antimicrobial defences of the small intestine. We found that the way in which cells are grown prior to formulation markedly affects sensitivity to acid and bile. Using a previously published stable storage formulation that maintained over 10% viability after 56 days storage at room temperature, we found dried samples of an attenuated S. typhimurium vaccine lost acid and bile resistance compared to the same bacteria taken from fresh culture. The stable formulation utilised osmotic preconditioning in defined medium plus elevated salt concentration to induce intracellular trehalose accumulation before drying. Dried bacteria grown in rich media without osmotic preconditioning showed more resistance to bile, but less stability during storage, suggesting a trade-off between bile resistance and stability. Further optimization is needed to produce the ultimate room-temperature stable oral live bacterial vaccine formulation.

Immunogenic properties of a recombinant fusion protein containing the C-terminal 19 kDa of Plasmodium falciparum merozoite surface protein-1 and the innate immunity agonist FliC flagellin of Salmonella Typhimurium

In a recent study, we demonstrated the immunogenic properties of a new malaria vaccine polypeptide based on a 19 kDa C-terminal fragment of the merozoite surface protein-1 (MSP1(19)) from Plasmodium vivax and an innate immunity agonist, the Salmonella enterica serovar Typhimurium flagellin (FliC). Herein, we tested whether the same strategy, based on the MSP1(19) component of the deadly malaria parasite Plasmodium falciparum, could also generate a fusion polypeptide with enhanced immunogenicity. The His(6)FliC-MSP1(19) fusion protein was expressed from a recombinant Escherichia coil and showed preserved in vitro TLR5-binding activity. In contrast to animals injected with His(6)MSP1(19), mice subcutaneously immunised with the recombinant His6FliC-MSP1(19) developed strong MSP1(19)-specific systemic antibody responses with a prevailing IgG1 subclass. Incorporation of other adjuvants, such as CpG ODN 1826, complete and incomplete Freund`s adjuvants or Quil-A, improved the IgG responses after the second, but not the third, immunising dose. It also resulted in a more balanced IgG subclass response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response, as determined by the detection of antigen-specific interferon-gamma secretion by immune spleen cells. MSP(19)-specific antibodies recognised not only the recombinant protein, but also the native protein expressed on the surface of P. falciparum parasites. Finally, sera from rabbits immunised with the fusion protein alone inhibited the in vitro growth of three different P. falciparum strains. In summary, these results extend our previous observations and further demonstrate that fusion of the innate immunity agonist FliC to Plasmodium antigens is a promising alternative to improve their immunogenicity. (c) 2010 Elsevier Ltd. All rights reserved.

A defective mutant of Salmonella enterica Serovar Gallinarum in cobalamin biosynthesis is avirulent in chickens

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efficacy of several Salmonella vaccination programs against experimental challenge with Salmonella gallinarum in commercial brown layer and broiler breeder hens

The protective effect of various Salmonella vaccines regimens against an experimental Salmonella Gallinarum challenge (SGNalr strain at 12 wk of age) was evaluated in two experiments. In Experiment 1 commercial brown layers were vaccinated according to one of the following programs: (i) two doses of a SE bacterin (Layermune SE; group 1); (ii) a first dose of a live SG9R vaccine (Cevac SG9R) followed by a SE bacterin (Layermune SE; group 2); (iii) one dose of each of two different multivalent inactivated vaccines containing SE cells (Corymune 4 & Corymune 7; group 3) or (iv) not vaccinated (group 4). In Experiment 2, broiler breeders were given the same vaccination treatments except for the group vaccinated with the multivalent vaccines. Overall, in both experiments, all vaccination schemes were effective in reducing mortality after challenge with a SG field strain. Primary vaccination with an initial dose of a live SG9R vaccine followed some weeks later by a dose of an inactivated SE bacterin was the most effective (p<0.05) vaccination program against mortality induced by field SG experimental challenge in both experiments. In conclusion, Salmonella vaccination programs containing SE bacterins alone or in combination with a live SG9R vaccine are effective in preventing mortality induced by infection of field SG. Nevertheless, it is important to emphasize that any vaccination program against any Salmonella serotype will only be effective if it is part of a sound and comprehensive biosecurity program designed for Salmonella control in poultry farms.

Efficacy of several vaccination programmes in commercial layer and broiler breeder hens against experimental challenge with Salmonella enterica serovar Enteritidis

Two experiments were performed to evaluate the protective effect of various vaccination combinations given at 5 and 9 weeks of age against experimental challenge with Salmonella enterica serovar Enteritidis ( SE) phage type 4 at 12 weeks of age. In Experiment 1, groups of commercial layers were vaccinated by one of the following programmes: Group 1, two doses of a SE bacterin (Layermune SE); Group 2, one dose of a live Salmonella enterica serovar Gallinarum vaccine (Cevac SG9R) followed by one dose of the SE bacterin; Group 3, one dose of each of two different multivalent inactivated vaccines containing SE cells (Corymune 4K and Corymune 7K; and Group 4, unvaccinated, challenged controls. In Experiment 2, groups of broiler breeders were vaccinated by the same programmes as Groups 1 and 2 above while Group 3 was an unvaccinated, challenged control group. All vaccination programmes and the challenge induced significant (P<0.05) seroconversion as measured by enzyme-linked immunosorbent assay. Overall, in both experiments, all vaccination schemes were significantly effective in reducing organ (spleen, liver and caeca) colonization by the challenge strain as well as reducing faecal excretion for at least 3 weeks. Vaccinated layers in Groups 1 and 2 and broiler breeders in Group 2 showed the greatest reduction in organ colonization and the least faecal excretion. In Experiment 1, layers vaccinated with multivalent inactivated vaccines containing a SE component (Group 3) were only moderately protected, indicating that such a vaccination programme may be useful in farms with good husbandry and housing conditions and low environmental infectious pressure by Salmonella.

Control of Salmonella enterica serovar Enteritidis in laying hens by inactivated Salmonella Enteritidis vaccines

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeito da vacina contra coccidiose sobre a colonização de Salmonella enteritidis em frangos de corte tratados com microbiota cecal anaeróbia

Avaliou-se o efeito da vacina contra coccidiose sobre a habilidade da microbiota cecal anaeróbia (MCA), administrada por diferentes vias (aves tratadas), em proteger pintos de corte da colonização por Salmonella enteritidis (Se). Utilizaram-se 120 aves assim divididas: grupo A, pulverização de MCA em aves vacinadas contra coccidiose e desafiadas com Se, grupo B, inoculação endoesofágica de MCA em aves vacinadas e desafiadas, grupo C, MCA na água de bebida de aves vacinadas e desafiadas, grupo D, aves não tratadas, vacinadas e desafiadas, grupo E, aves não tratadas, não vacinadas e desafiadas e, grupo F, aves não tratadas, não vacinadas e não desafiadas (controle negativo). Utilizaram-se como parâmetros a colonização do trato digestivo por Se e sua presença nas fezes, bem como o peso corporal das aves, avaliados aos dois, sete e 12 dias após o desafio. Nas aves tratadas com MCA, especialmente por meio de pulverização e inoculação endoesofágica, a colonização do ceco por Se e sua presença nas fezes foram menores, mostrando que a ação da MCA contra a colonização de ceco e excreção fecal não foi afetada pelo uso da vacina contra coccidiose em pintos de corte e que a associação MCA/vacina contra coccidiose pode ser utilizada sem que haja comprometimento na eficácia da MCA. Nos grupos que não receberam MCA, vacinados ou não contra coccidiose, houve aumento da colonização cecal, bem como excreção fecal da amostra desafio. O ceco foi o local de maior presença e persistência da Se. O resultado de administração de MCA pela água de bebida não foi tão eficiente, mas somente este tratamento resultou em peso corporal das aves significativamente superior ao das aves do grupo não tratado, não vacinado e desafiado, indicando que a presença de salmonelas paratifóides não interfere na produtividade de frangos de corte. Não se observou diferença no peso das aves dos grupos D (vacinados contra coccidiose) e E (não vacinadas) desafiadas com Se, demonstrando que a vacina não influenciou negativamente o ganho de peso das aves desafiadas com Salmonela enteriditis.

Experimental infection of commercial layers with wild or attenuated Salmonella Gallinarum mutant strains: Anatomic pathology, total blood cell count and serum protein levels

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Utilização de um mutante atenuado de Salmonella enterica subesp. enterica sorovar GALLINARUM cobS cbiA para proteção de aves contra a infecção por Salmonella enterica subesp. enterica sorovares GALLINARUM E ENTERITIDIS

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Infecção experimental de aves de postura (Gallus gallus domesticus) por cepas de Salmonella enterica sorovar Gallinarum (SG), SGNalr SGcobS e SGcobScbiA: Anatomopatologia, hemograma e perfil bioquímico sérico

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Resposta imune celular e humoral em aves (Gallus gallus) vacinadas, antes e após o desafio com Salmonella enteritidis

Pós-graduação em Medicina Veterinária - FCAV

Efeito da inoculação de probiótico in ovo sobre a morfometria intestinal e controle de salmonella enteritidis

Pós-graduação em Zootecnia - FMVZ

Immunomodulatory activity and control of Salmonella Enteritidis colonization in the intestinal tract of chickens by Lactobacillus based probiotic

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)