Bacteriocins are antimicrobial protein produced by certain Gram positive and negative bacteria as a defense mechanism against closely related bacteria competing for the same nutrient or in the same niche. The competition for the same nutrient is supported by the fact that bacteriocins have narrow range of effect and only likely to be effective against closely related bacteria for the same scares resources hence a bacteriocin produced by a Gram positive bacteria will be active against a Gram positive pathogens and a bacteriocin produced by a Gram negative bacteria will be active against Gram negative pathogens. This is due to the difference in cell wall composition, they are either bacteriocidal or bacteriostatic Bacteriocins have been used for thousands of years for food preservation unknowingly to man, they are considered advantageous not only to the producing bacteria, but it's now been used by the food industry as a tool to control both spoilage and pathogenic bacteria in food, in a natural manner which is acceptable to the consumer. With a lot of research been carried out on bacteriocins produced by Gram positive bacteria, antagonist to Gram positive food borne pathogens, little is known about bacteriocins produced by Gram negative bacteria which would be active against Gram negative food borne pathogens that predominate in produce. The objective of my research therefore is to screen for antimicrobial antagonist to Gram negative food borne pathogens (Escherichia coli O157:H7 and Salmonella Enteritidis) from produce, to determine an appropriate screening method, to carry out a preliminary characterization of antagonist discovered and also to determine antimicrobial spectrum of antagonist found. Lettuce was screened for antimicrobial antagonist against Gram negative pathogen (Escherichia coli O157:H7 and Salmonella Enteritidis) which were used as indicator strains With over 5000 colonies screen, 1 colony (Serratia plymuthica) was discovered to be antagonistic against these indicator strain. Further screening of cell free extract using the spot test method showed that extract from Serratia plymuthica grown alone in TSBYE showed antagonist activity against indicator strain with a little clearing on the spot of extract dropped. But extract of a co-culture of Serratia plymuthica and either Escherichia coli O157:H7 or Salmonella Enteritidis showed a more obvious clearing around spotted zone, which further indicates antagonism against indicator strains. Preliminary heat test indicates antagonist compound to be heat stable at 60oC for 30mins, 100oC for 30minutes and 60mins and 121oC for 20minites, and antagonist compound possessed antagonist activity against other strains of Escherichia coli when tested.

No description available.

Contaminated poultry and egg products are major sources of Salmonella enterica serovar Enteritidis (S. enteritidis, SE) infections in humans. Colonization of SE in chicken reproductive tract results in the production of contaminated commercial shell-eggs and fertilized hatchery eggs. The complex pathogen-host interactions during SE colonization of chicken reproductive tract are largely unknown. This study was aimed at determining the pathogenic roles of the type three secretion systems (TTSS-1 and TTSS-2) in SE infection of chicken oviduct epithelial cells (COEC). A series of SE strains carrying mutations in the genes encoding structure or effector proteins of TTSS-1 and TTSS-2 were constructed. The invasiveness and intracellular survival rate of each SE strain as well as the host innate immune responses induced by the infections were evaluated. The results demonstrate that both TTSS-1 and TTSS-2 are required by SE to invade COEC which involve genes encoding effector proteins SipA, SopB, SopE2, and PipB. In addition to their involvement in host cell invasion, sipA and sipB are also necessary for the survival or replication of SE inside COEC. Inactivation of TTSS-2 genes (ssaV and pipB) resulted in an enhanced bacterial proliferation inside COEC. The data from this study also show that SE infection triggers pro-inflammatory responses in COEC and TTSS-1 is involved in the expression of iNOS and IL-8, a CXC chemokine. TTSS-1 and TTSS-2 are not necessary for induction of K203, MIP-1β, and IL-10 or suppression of TGF-β3 in COEC.

No description available.

La microcine J25 (MccJ25), une bactériocine en forme de lasso synthétisée par Escherichia coli et ayant une activité inhibitrice contre Salmonella, a été produite et purifiée d’un surnageant d’E. coli pTUC202. La stabilité et l’activité biologique de cette bactériocine au niveau gastro-intestinal, ainsi que son impact sur l’équilibre du microbiote colique, ont été étudiés à l’aide d’un système de fermentation en continu avec cellules immobilisées reproduisant les conditions physiologiques et microbiologiques du côlon. L’analyse par qPCR du microbiote colique n’a révélé aucun effet significatif sur l’équilibre du microbiote de la MccJ25 lorsqu’elle est ajoutée à 0,4, 2 et 5 μM. Des tests d’activité antibactérienne ont démontré que la MccJ25 reste active contre Salmonella enteritidis dans les conditions coliques testées. Ces résultats suggèrent un fort potentiel d’utilisation de la MccJ25 comme alternative aux antibiotiques aussi bien chez l’animal que chez l’humain. / Microcin J25 (MccJ25) is a lasso-shaped bacteriocin with an antibacterial activity against Salmonella. This bacteriocin was produced and purified from the supernatant of Escherichia coli pTUC202. Its stability and biological activity in the gastrointestinal tract as well as its impact on the colonic microbiota equilibrium were studied using a continuous fermentation system with immobilized fecal microbiota simulating the physiological and microbiological conditions of the colon. Analysis of colonic microbiota by qPCR revealed no significant effect of MccJ25 on microbiota equilibrium at concentrations of 0.4, 2 et 5 μM. Antibacterial activity assays demonstrated that MccJ25 remains active against Salmonella enteritidis in the tested colonic conditions. These results are highly promising for the use of MccJ25 as an alternative to antibiotics in animals as well as in humans.

Les infections à Salmonella Enteritidis chez les humains sont associées à la consommation d’œufs ou d’ovoproduits contaminés. La vaccination est un outil utilisé pour diminuer les risques d’infection à SE chez la volaille, mais avec des résultats variables. Au Canada deux bactérines, MBL SE4C et Layermune, sont couramment utilisées pour lutter contre SE. Cependant, leur efficacité n’a pas été complètement déterminée chez les poules pondeuses plus âgées. Par ailleurs, la capacité de ces vaccins à prévenir la transmission verticale et horizontale n’a pas encore été étudiée. L’objectif principal de cette étude était d’évaluer l’effet des deux bactérines sur la réponse immunitaire chez les poules pondeuses, de vérifier la protection conférée par ces vaccins contre l’infection expérimentale à SE, et d’identifier des protéines immunogènes afin de développer un vaccin sous-unitaire. Les oiseaux ont été vaccinés avec deux protocoles d’immunisation en cours d’élevage (soit à 12 et 18, ou à 16 semaines d’âge). Le groupe contrôle a été injecté avec la solution saline. Les oiseaux ont été inoculés per os avec 2 x 109 CFU de la souche SE lysotype 4 à 55 ou à 65 semaines d’âge. Les anticorps (IgG et IgA) ont été mesurés à différents temps avec un ELISA maison en utilisant l’antigène entier de SE. La phagocytose, flambée oxydative, les populations des splénocytes B et T ont été analysées en utilisant la cytométrie en flux. Les signes cliniques, l’excrétion fécale, la contamination des jaunes d’œufs et l’invasion des salmonelles dans les organes ont été étudiés pour évaluer l’efficacité de protection. La transmission horizontale a aussi été étudiée en évaluant l’infection à SE chez les oiseaux mis en contact avec les oiseaux inoculés. Les protéines immunogènes ont été identifiées par SDS-PAGE et Western blot à l’aide d’antisérums prélevés suite à la vaccination et/ou à l’infection expérimentale/naturelle, puis caractérisées par la spectrométrie de masse. Le protocole de vaccination avec deux immunisations a généré un niveau élevé de séroconversion à partir de 3 jusqu’à 32-34 semaines post-vaccination par rapport à celui avec une seule immunisation (p < 0.02), mais il n’y avait plus de différence entre les groupes à 54 et 64 semaines d’âge. Il n’y a pas eu de corrélation entre les niveaux d’IgG et les taux d’isolement des salmonelles dans les organes et des jaunes d’œuf. La production des IgA n’a été observée que chez les oiseaux vaccinés avec 2 injections de MBL SE4C (p ≤ 0.04). Après l’infection expérimentale, la production des IgA a été significativement plus élevée aux jours 1 et 7 p.i dans l’oviducte des oiseaux vaccinés (sauf pour le groupe vacciné avec 2 injections de Layermune) par comparaison avec le groupe contrôle (p ≤ 0.03). Seule la bactérine MBL SE4C a eu un effet protecteur contre la contamination des jaunes d’œuf chez les oiseaux infectés. Ce vaccin réduit partiellement en utilisant deux immunisations, le taux d’excrétion fécale des salmonelles chez les oiseaux inoculés et les oiseaux horizontalement infectés (p ≤ 0.02). Cinq des protéines identifiées par la spectrométrie de masse sont considérées comme des protéines potentiellement candidates pour une étude plus approfondie de leur immonogénicité: Lipoamide dehydrogenase, Enolase (2-phosphoglycerate dehydratase) (2-phospho-D-glycerate hydro-lyase), Elongation factor Tu (EF-Tu), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) et DNA protection during starvation protein. En général, les bactérines ont induit une immunité humorale (IgG et IgA) chez les poules pondeuses. Cette réponse immunitaire a protégé partiellement les oiseaux quant à l’élimination des salmonelles, la contamination des jaunes d’œuf, ainsi que la transmission horizontale. Dans cette étude, la bactérine MBL SE4C (avec deux immunisations) s’est montrée plus efficace pour protéger les oiseaux que la bactérine Layermune. Nos résultats apportent des informations objectives et complémentaires sur le potentiel de deux bactérines pour lutter contre SE chez les poules pondeuses. Étant donné la protection partielle obtenue en utilisant ces vaccins, l’identification des antigènes immunogènes a permis de sélectionner des protéines spécifiques pour l’élaboration éventuelle d’un vaccin plus efficace contre SE chez les volailles. / Contaminated eggs and egg products have been associated with outbreaks of human Salmonella Enteritidis (SE) infections. Killed bacteria (bacterins) have been used to control Salmonella infections in poultry but variation in the conferred protection has been observed. In Canada the bacterins MBL SE4C and Layermune are currently used to control SE. However, their efficacy in protecting older layers has not been fully determined. Furthermore, the capacity of these bacterins to prevent vertical and horizontal transmissions has not yet been investigated. The main objectives of this study were to evaluate the effect of two available commercial bacterins on the immune response of laying hens, to verify the protection conferred by these vaccines against SE challenge and to identify immunogenic proteins to develop an oral subunit vaccine. Laying hens were vaccinated with two immunization schedules prior to the lay cycle (either at 12 and 18, or 16 weeks of age). The control group was injected with a saline solution. Laying hens were later inoculated per os with 2 x 109 CFU of SE PT4 strain either at 55 or 65 weeks of age. Serum IgG and mucosal IgA antibodies were measured with an in-house SE whole cell antigen ELISA. The phagocytosis, oxidative burst, splenic T and B cells populations were analyzed using flow cytometry. Clinical signs, fecal shedding, egg yolks contamination and organ invasion by SE were assessed to evaluate vaccine protection. Potential horizontal transmission from inoculated laying hens to non-inoculated laying hens, housed in the same isolator unit, was also evaluated. Immunogenic proteins were identified by SDS-PAGE and Western blot with sampled antisera during vaccination and/or infection of poultry with SE and then subjected to mass spectrometry. The vaccination protocol with two immunizations showed a higher seroconversion level than the single vaccination at 3 until 32-34 weeks post vaccination (p < 0.02) but no difference before challenge (54 and 64 old weeks). There was no relationship between high IgG level and SE isolation rates in organs and egg yolks. Only the MBL SE4C vaccine elicited IgA antibody production at 3 weeks post vaccination in both immunization protocols (p ≤ 0.04). Significant higher mucosal IgA levels were observed at day 1 and 7 post challenge in oviduct of vaccinated birds (except for the twice vaccinated Layermune group) compared to the control group (p ≤ 0.03). Humoral efficacy to protect from SE contamination of egg yolk was only observed in MBL SE4C vaccinated group and only this bacterin administered twice reduced SE shedding rate in inoculated birds and their exposed cagemates (p ≤ 0.02). A set of 5 proteins were considered as putative protein candidates to further detailed study on their immunogenicity: Lipoamide dehydrogenase; Enolase (2-phosphoglycerate dehydratase) (2-phospho-D-glycerate hydro-lyase); Elongation factor Tu (EF-Tu); Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and DNA protection during starvation protein. Overall, the commercial bacterins induced humoral immunity (IgG and IgA antibodies) in laying hens. This immune response partially protected for SE clearance, egg yolks contamination as well as horizontal transmission. In this study, MBL SE4C bacterin appeared to be more efficient in comparison to Layermune for protection of hens with a vaccination protocol comprising two immunizations. Our results provide additional and objective information on the potential of these vaccines for the control of SE in laying hens. Considering the partial protection achieved with the use of these bacterins, the identification of immunogenic antigens could help in the selection of specific proteins to elaborate a more efficient vaccine against SE in poultry.

Salmonella é um dos principais agentes de enfermidades transmitidas por alimentos em diversos países, sendo a carne de frango um dos principais veículos envolvidos em surtos. O Brasil vem se destacando como um dos maiores exportadores mundiais deste alimento. O ambiente de criação das aves é apontado como um importante foco de infecção das aves e o ambiente industrial de abate e processamento é importante na disseminação deste ·microrganismo. Na busca pela produção de alimentos seguros do ponto de vista microbiológico, uma das ferramentas utilizadas é a subtipagem de microrganismos isolados ao longo da cadeia de produção, que permite determinar rotas de contaminação do produto final. Os objetivos deste trabalho são: o estudo da disseminação dos subtipos de Salmonella Enteritidis nas várias etapas de uma cadeia de produção industrial de carne de frango, empregando-se diversos métodos de subtipagem e a determinação da resistência a antimicrobianos destas cepas. 108 isolados de Salmonella Enteritidis dos fagotipos PT1, PT4 e PT7a foram obtidos nos anos de 2002 e 2003, a partir de amostras ambientais e de frango relativas a sete sub-regiões de uma cadeia produtiva industrial avícola. Os perfis de resistência destes isolados foram determinados frente a antimicrobianos de uso humano e veterinário e eles foram submetidos a subtipagem por PFGE, RAPO, ribotipagem e PCR-ribotipagem. Foram detectados 21 perfis de resistência diferentes, com 6,5% das cepas sensíveis a todas as drogas, 33,3% resistentes a um ou dois antimicrobianos e 83,3% apresentando resistência intermediária a até quatro deles. Os níveis relativamente elevados de resistência são preocupantes e a diminuição da pressão seletiva deve ser um objetivo para os produtores de aves. De modo geral, a subtipagem permitiu separar as cepas em 13 genótipos, com elevada similaridade entre si. Porém, a maior parte das cepas (69,4%) pertenceu a apenas três deles, que foram encontrados ao longo de toda a cadeia produtiva. A ribotipagem foi o método que apresentou o melhor poder discriminatório (D = 0,701), porém nem todas as cepas foram tipáveis por este método. Não foram encontradas correlações entre os perfis de resistência a antimicrobianos e fagotipos, nem entre genótipos e fagotipos. Porém, dois genótipos proximamente correlacionados e predominantemente encontrados em uma sub-região reuniram apenas cepas com resistência intermediária ou resistentes exclusivamente à furazolidona. A similaridade elevada entre os genótipos evidencia a origem clonal das cepas. / Salmonella is one of the most important foodborne disease agents all over the world, and chicken is recognized as an important vehicle of the infection. Chicken production in Brazil has increased in the last couple of years and the country is now ranked 2nd as producer/exporter of this commodity. For this reason there is an increased concern over the safety of these goods. This study deals with the dissemination, antimicrobial resistance, and genetic characterization of S. Enteritidis strains isolated from an industrial chicken production chain. 108 isolates, phagetypes PT1, PT4 and PT7a, were obtained at different steps of the commercial production from farm to frozen cuts, and the broilers were from different producers supplying the same processing plant. Tests for susceptibility to 12 human and veterinary antimicrobial agents were performed. The strains were also typed by PFGE, RAPO, ribotyping, and PCR-ribotyping. 6.5% of the strains were susceptible to the 12 drugs tested and 33.3% were resistant to 1 or 2 of them. Intermediate resistance to up to 4 agents was observed in 83.3% of the isolates. Combining all the typing methods allowed the division of the strains in 13 genotypes with elevated degree of similarity. However, 69.4% of the strains belonged to 3 main phagetypes spread along the production chain. There was no correlation between phagetypes and genotypes, or phagetypes and resistance profiles. However, most strains from one sub-region were from 2 genotypes and showed intermediate resistance to, or were resistant to furazolidone. The high degree of similarity amongst the genotypes indicates the clonal origin of the strains. The relatively high resistance to antimicrobial agents is a cause of concern and trying to diminish the selective pressure has to be a goal for broiler producers.

La présence de bactéries dans le sang, un milieu normalement stérile, peut avoir des conséquences graves voire fatales pour l’organisme atteint. Afin de diagnostiquer au plus tôt cette infection, appelée bactériémie, et ainsi administrer le traitement adéquat, il est nécessaire d’identifier les microorganismes isolés à partir du sang. Mais, la nature complexe de ce fluide biologique, associée à la faible charge bactérienne, parfois inférieure à 1 UFC par millilitre de sang ont des conséquences sur les méthodes pouvant être utilisées pour l’identification des bactéries. La plupart d’entre elles ont donc recours à une première étape, l’hémoculture, au cours de laquelle les microorganismes présents dans le prélèvement sanguin de volume important (20-30 mL) vont se multiplier. Leur croissance est facilitée par la dilution du sang dans des milieux de culture dédiés à cette étape particulière. C’est seulement ensuite que l’identification peut débuter. Elle nécessite encore entre 2 et 48 heures et parfois plus, selon les moyens à disposition et les microorganismes impliqués. Réduire considérablement le temps nécessaire à l’identification aurait pourtant des retombées bénéfiques à l’échelle du patient mais aussi plus globalement en réduisant les coûts associés à cette infection et en limitant la pression de sélection exercée par l’emploi d’antibiotiques à large spectre.Au cours de ce travail, l’évaluation d’une stratégie basée sur l’identification des bactéries lors de leur multiplication dans le milieu d’hémoculture est donc proposée. Elle repose sur l’observation en temps réel et sans marquage par Résonance Plasmonique de Surface par imagerie (SPRi) des interactions entre les bactéries et des ligands déposés à la surface d’un capteur. Dans un premier temps, des ligands alternatifs aux anticorps parmi lesquels figurent les aptamères, des protéines de l’immunité innée et la vancomycine sont testés. Suite à cette étude, les anticorps ont été retenus pour poursuivre ce travail. Leur emploi n’est cependant pas dénué de difficultés lorsqu’il s’agit de détecter spécifiquement Staphylococcus aureus, choisi comme l’un des modèles expérimentaux. En effet, la présence de protéine A chez cette bactérie est à l’origine d’interférences sur les immunoglobulines. Différentes stratégies pour s’affranchir de ces effets ont été évaluées, comme le clivage enzymatique des anticorps ou l’emploi d’anticorps de poule pour lesquels la protéine A n’a pas d’affinité. Ces essais aboutissent à des résultats encourageants en milieu de culture simple. L’ajout de sérum humain au milieu a soulevé de nouveaux problèmes pour la détection de cette bactérie. Les résultats montrent qu’en interagissant avec des constituants de l’échantillon sanguin, dont les anticorps, S. aureus devient indétectable par une biopuce à anticorps. Une discussion des moyens possibles pour lever cette inhibition est ensuite proposée. Des expériences de détection d’une autre bactérie, Salmonella enterica sérovar Enteritidis pour laquelle nous disposons d’un anticorps hautement affin et spécifique ont alors été entreprises afin de conclure sur l’employabilité du dispositif dans des conditions proches d’une hémoculture. Des interférences affectant différentiellement les anticorps selon leur point isoélectrique ont ainsi été mises en évidences et l’implication de l’anticoagulant (polyanéthole sulfonate de sodium, SPS) présent dans les milieux d’hémoculture a été démontrée. La résolution partielle de ce problème a finalement permis la détection de 1 UFC.mL-1 de sang dans 32 mL au total démontrant ainsi la possibilité de détecter spécifiquement une bactérie dans des conditions proches d’une hémoculture. / The presence of bacteria in the blood, a normally sterile environment, can cause dramatic consequences for an organism. In order to diagnose this infection, called bacteremia, the identification of the microorganism present in blood must be performed. Furthermore, proper diagnosis enables the administration of a suitable antibiotic therapy. Blood complexity as well as the low bacterial load, usually lower than 1 CFU.mL-1, make the diagnosis of this infection quite challenging. Indeed, most identification methods begin only after the blood culture turns positive due to their insufficient sensitivity. For this they require incubation of a large blood sample volume (20 – 30 mL) in specific culture media that allows bacterial growth above their detection limit. Therefore, its increases considerably the time of diagnosis, which usually takes between 2 and 48 hours and sometimes even more time after blood culture positivity depending on the method and the microorganism present in blood. A reduction of the time required for identification would have a positive impact for both the patient and the healthcare systems by reducing selective pressure on resistant bacteria and hospitalization costs by giving proper treatment faster.In this work, the evaluation of a new strategy based on the identification of bacteria during their multiplication in the blood culture is presented. This method is based on Surface Plasmon Resonance imaging (SPRi) which enables real time and label-free measurements of interactions occurring between bacteria and specific probes. Alternative ligands like aptamers, innate immune proteins and vancomycin have been tested. Following this study antibodies have been chosen as the major specific probes in this work. Nonetheless, the presence of the staphylococcal protein A leads to false-positive results in all immunoglobulin G (IgG). Enzymatic cleavage to remove the constant fragment of antibody where protein A interacts and the use of chicken antibodies (IgY) for which protein A has no affinity have been evaluated. Both methods allow to get rid of protein A interactions in pure culture media. But the presence of human serum in the media results in the total loss of signal. Our results show that interactions between blood components and staphylococcal proteins exposed at the bacterial surface, including the interactions between protein A and circulating antibodies, are responsible for this phenomenon. Solutions to alleviate this inhibition are discussed and tested. Detection experiments of another bacterial model, Salmonella enterica serovar Enteritidis in blood culture media are presented. The crucial role played by the anticoagulant Sodium Polyanethole Sulfonate in non-specific interactions on antibodies is demonstrated. These interactions leading to a total loss of specificity for some antibodies are influenced by the isoelectric point (pI) of the probes which interact with this anionic compound and then attract blood components. After the partial resolution of this issue, we show the feasibility of detecting less than one bacteria per blood milliliter in a total volume of 32 milliliters, conditions close to real blood culture.

Salmonella Enteritidis (SE) é um enteropatógeno de grande preocupação para indústria de alimentos, principalmente de produtos que não podem ser submetidos a tratamento térmico, como as saladas à base de maionese, freqüentemente envolvidas em surtos de salmonelose. O uso de antimicrobianos naturais nesses produtos pode ser um método alternativo para o controle de SE. Assim, o objetivo deste trabalho foi avaliar o efeito antimicrobiano de óleo essencial de orégano, EDTA e nisina individualmente e a combinação de nisina com EDTA e nisina com óleo essencial de orégano no controle da multiplicação SE em salada de legumes com maionese. A atividade inibitória foi inicialmente avaliada in vitro e posteriormente em salada preparada com legumes, experimentalmente contaminados com SE, misturados à maionese, na concentração de 103 UFC/g, e armazenados em refrigeração (8°C) e em temperatura ambiente (30°C). Os resultados da avaliação in vitro indicaram que o OEO usado individualmente tem melhor efeito antimicrobiano contra SE do que quando empregado em combinação com nisina. Nem a nisina nem o EDTA, quando testados isoladamente ou combinados apresentaram efeito sobre SE. Na salada de legumes com maionese preparada artesanalmente, a presença de 0,2% de OEO resultou em redução na contagem de SE, constituindo-se em uma barreira adicional para a multiplicação do patógeno nesse produto. A análise sensorial da salada de legumes com maionese contendo 0,2% de OEO indicou que houve 95% de aceitação do aroma, 93% de aceitação do sabor e 74% de intenção de compra. A análise sensorial da maionese contendo 0,5% ou 1% de OEO indicou a inviabilidade de se aumentar a concentração do OEO na maionese como alternativa para melhorar o efeito antimicrobiano. / Salmonella Enteritidis (SE) is an enteropathogen relevant for the food industry, especially in foods that do not require heat treatment before consumption such as salads prepared with mayonnaise, frequently associated to outbreaks of salmonellosis. Natural antimicrobials can be used as an alternative procedure to control SE in these foods. The aim of the present study was to evaluate the effect of origanum essential oil (OEO), nisin and EDTA, used individually and in combination, on the growth of SE in mayonnaise and in mayonnaise-based legume salad. The inhibitory activity was evaluated in vitro, in home made mayonnaise and in mayonnaise-based legume salad experimentally contaminated with SE at 103 CFU/g, stored under refrigeration (8°C) and at room temperature (30°C). The results of the in vitro tests indicated that a better antimicrobial effect of OEO on SE was achieved when OEO was used individually than when used combined with nisin or EDTA. Nor nisin nor EDTA, used individually, presented any effect against SE. The presence of OEO in mayonnaise did not add any additional effect to the antimicrobial activity of the intrinsec parameters in this food. In mayonnaise-based legume salad, 0.2% of OEO caused a reduction in the counts of SE, and was an additional hurdle for the growth of the pathogen in this product. The sensorial testing of mayonnaise-based legume salad containing 0.2% OEO indicated an acceptance of 95% for the aroma and 93% for the taste and 74% of purchasing intention. The sensorial testing of mayonnaise containing 0.5% or 1% OEO indicated that the increase in the content of OEO to increase the antimicrobial activity is not feasible.

Les infections à Salmonella Enteritidis chez les humains sont associées à la consommation d’œufs ou d’ovoproduits contaminés. La vaccination est un outil utilisé pour diminuer les risques d’infection à SE chez la volaille, mais avec des résultats variables. Au Canada deux bactérines, MBL SE4C et Layermune, sont couramment utilisées pour lutter contre SE. Cependant, leur efficacité n’a pas été complètement déterminée chez les poules pondeuses plus âgées. Par ailleurs, la capacité de ces vaccins à prévenir la transmission verticale et horizontale n’a pas encore été étudiée. L’objectif principal de cette étude était d’évaluer l’effet des deux bactérines sur la réponse immunitaire chez les poules pondeuses, de vérifier la protection conférée par ces vaccins contre l’infection expérimentale à SE, et d’identifier des protéines immunogènes afin de développer un vaccin sous-unitaire. Les oiseaux ont été vaccinés avec deux protocoles d’immunisation en cours d’élevage (soit à 12 et 18, ou à 16 semaines d’âge). Le groupe contrôle a été injecté avec la solution saline. Les oiseaux ont été inoculés per os avec 2 x 109 CFU de la souche SE lysotype 4 à 55 ou à 65 semaines d’âge. Les anticorps (IgG et IgA) ont été mesurés à différents temps avec un ELISA maison en utilisant l’antigène entier de SE. La phagocytose, flambée oxydative, les populations des splénocytes B et T ont été analysées en utilisant la cytométrie en flux. Les signes cliniques, l’excrétion fécale, la contamination des jaunes d’œufs et l’invasion des salmonelles dans les organes ont été étudiés pour évaluer l’efficacité de protection. La transmission horizontale a aussi été étudiée en évaluant l’infection à SE chez les oiseaux mis en contact avec les oiseaux inoculés. Les protéines immunogènes ont été identifiées par SDS-PAGE et Western blot à l’aide d’antisérums prélevés suite à la vaccination et/ou à l’infection expérimentale/naturelle, puis caractérisées par la spectrométrie de masse. Le protocole de vaccination avec deux immunisations a généré un niveau élevé de séroconversion à partir de 3 jusqu’à 32-34 semaines post-vaccination par rapport à celui avec une seule immunisation (p < 0.02), mais il n’y avait plus de différence entre les groupes à 54 et 64 semaines d’âge. Il n’y a pas eu de corrélation entre les niveaux d’IgG et les taux d’isolement des salmonelles dans les organes et des jaunes d’œuf. La production des IgA n’a été observée que chez les oiseaux vaccinés avec 2 injections de MBL SE4C (p ≤ 0.04). Après l’infection expérimentale, la production des IgA a été significativement plus élevée aux jours 1 et 7 p.i dans l’oviducte des oiseaux vaccinés (sauf pour le groupe vacciné avec 2 injections de Layermune) par comparaison avec le groupe contrôle (p ≤ 0.03). Seule la bactérine MBL SE4C a eu un effet protecteur contre la contamination des jaunes d’œuf chez les oiseaux infectés. Ce vaccin réduit partiellement en utilisant deux immunisations, le taux d’excrétion fécale des salmonelles chez les oiseaux inoculés et les oiseaux horizontalement infectés (p ≤ 0.02). Cinq des protéines identifiées par la spectrométrie de masse sont considérées comme des protéines potentiellement candidates pour une étude plus approfondie de leur immonogénicité: Lipoamide dehydrogenase, Enolase (2-phosphoglycerate dehydratase) (2-phospho-D-glycerate hydro-lyase), Elongation factor Tu (EF-Tu), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) et DNA protection during starvation protein. En général, les bactérines ont induit une immunité humorale (IgG et IgA) chez les poules pondeuses. Cette réponse immunitaire a protégé partiellement les oiseaux quant à l’élimination des salmonelles, la contamination des jaunes d’œuf, ainsi que la transmission horizontale. Dans cette étude, la bactérine MBL SE4C (avec deux immunisations) s’est montrée plus efficace pour protéger les oiseaux que la bactérine Layermune. Nos résultats apportent des informations objectives et complémentaires sur le potentiel de deux bactérines pour lutter contre SE chez les poules pondeuses. Étant donné la protection partielle obtenue en utilisant ces vaccins, l’identification des antigènes immunogènes a permis de sélectionner des protéines spécifiques pour l’élaboration éventuelle d’un vaccin plus efficace contre SE chez les volailles. / Contaminated eggs and egg products have been associated with outbreaks of human Salmonella Enteritidis (SE) infections. Killed bacteria (bacterins) have been used to control Salmonella infections in poultry but variation in the conferred protection has been observed. In Canada the bacterins MBL SE4C and Layermune are currently used to control SE. However, their efficacy in protecting older layers has not been fully determined. Furthermore, the capacity of these bacterins to prevent vertical and horizontal transmissions has not yet been investigated. The main objectives of this study were to evaluate the effect of two available commercial bacterins on the immune response of laying hens, to verify the protection conferred by these vaccines against SE challenge and to identify immunogenic proteins to develop an oral subunit vaccine. Laying hens were vaccinated with two immunization schedules prior to the lay cycle (either at 12 and 18, or 16 weeks of age). The control group was injected with a saline solution. Laying hens were later inoculated per os with 2 x 109 CFU of SE PT4 strain either at 55 or 65 weeks of age. Serum IgG and mucosal IgA antibodies were measured with an in-house SE whole cell antigen ELISA. The phagocytosis, oxidative burst, splenic T and B cells populations were analyzed using flow cytometry. Clinical signs, fecal shedding, egg yolks contamination and organ invasion by SE were assessed to evaluate vaccine protection. Potential horizontal transmission from inoculated laying hens to non-inoculated laying hens, housed in the same isolator unit, was also evaluated. Immunogenic proteins were identified by SDS-PAGE and Western blot with sampled antisera during vaccination and/or infection of poultry with SE and then subjected to mass spectrometry. The vaccination protocol with two immunizations showed a higher seroconversion level than the single vaccination at 3 until 32-34 weeks post vaccination (p < 0.02) but no difference before challenge (54 and 64 old weeks). There was no relationship between high IgG level and SE isolation rates in organs and egg yolks. Only the MBL SE4C vaccine elicited IgA antibody production at 3 weeks post vaccination in both immunization protocols (p ≤ 0.04). Significant higher mucosal IgA levels were observed at day 1 and 7 post challenge in oviduct of vaccinated birds (except for the twice vaccinated Layermune group) compared to the control group (p ≤ 0.03). Humoral efficacy to protect from SE contamination of egg yolk was only observed in MBL SE4C vaccinated group and only this bacterin administered twice reduced SE shedding rate in inoculated birds and their exposed cagemates (p ≤ 0.02). A set of 5 proteins were considered as putative protein candidates to further detailed study on their immunogenicity: Lipoamide dehydrogenase; Enolase (2-phosphoglycerate dehydratase) (2-phospho-D-glycerate hydro-lyase); Elongation factor Tu (EF-Tu); Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and DNA protection during starvation protein. Overall, the commercial bacterins induced humoral immunity (IgG and IgA antibodies) in laying hens. This immune response partially protected for SE clearance, egg yolks contamination as well as horizontal transmission. In this study, MBL SE4C bacterin appeared to be more efficient in comparison to Layermune for protection of hens with a vaccination protocol comprising two immunizations. Our results provide additional and objective information on the potential of these vaccines for the control of SE in laying hens. Considering the partial protection achieved with the use of these bacterins, the identification of immunogenic antigens could help in the selection of specific proteins to elaborate a more efficient vaccine against SE in poultry.