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This study was conducted to evaluate the performance of a screening protocol to detect and isolate mcr-positive Escherichia coli and Salmonella spp. from animal caecal content and meat samples. We used a multicentre approach involving 12 laboratories from nine European countries. All participants applied the same methodology combining a multiplex PCR performed on DNA extracted from a pre-enrichment step, followed by a selective culture step on three commercially available chromogenic agar plates. The test panel was composed of two negative samples and four samples artificially contaminated with E. coli and Salmonella spp. respectively harbouring mcr-1 or mcr-3 and mcr-4 or mcr-5 genes. PCR screening resulted in a specificity of 100% and a sensitivity of 83%. Sensitivity of each agar medium to detect mcr-positive colistin-resistant E. coli or Salmonella spp. strains was 86% for CHROMID(R)Colistin R, 75% for CHROMagarTM COL-APSE and 70% for COLISTIGRAM. This combined method was effective to detect and isolate most of the E. coli or Salmonella spp. strains harbouring different mcr genes from food-producing animals and food products and might thus be used as a harmonized protocol for the screening of mcr genes in food-producing animals and food products in Europe.

Salmonella Enteritidis is the most prevalent food-borne pathogen associated with egg-related outbreaks in the European Union. During egg colonization, S. Enteritidis must resist the powerful anti-bacterial activities of egg white (EW) and overcome ovotransferrin-imposed iron-restriction (the most important anti-bacterial mechanism of EW). Many pathogens respond to iron restriction by secreting iron-chelating chemicals called siderophores but EW contains a siderophore-sequestering "lipocalin" protein (Ex-FABP) that is predicted to limit the usefulness of siderophores in EW. S. Enteritidis produces two siderophores: enterobactin, which is strongly bound by Ex-FABP; and the di-glucosylated enterobactin-derivative, salmochelin (a so-called "stealth" siderophore), which is not recognized by Ex-FABP. Thus, production of salmochelin may allow S. Enteritidis to escape Ex-FABP-mediated growth inhibition under iron restriction although it is unclear whether its EW concentration is sufficient to inhibit pathogens. Further, two other lipocalins (Cal-γ and α-1-ovoglycoprotein) are found in EW but their siderophore sequestration potential remains unexplored. In addition, the effect of EW lipocalins on the major EW pathogen, S. Enteritidis, has yet to be reported. We overexpressed and purified the three lipocalins of EW and investigated their ability to interact with the siderophores of S. Enteritidis, as well as their EW concentrations. The results show that Ex-FABP is present in EW at concentrations (5.1 μM) sufficient to inhibit growth of a salmochelin-deficient S. Enteritidis mutant under iron restriction but has little impact on the salmochelin-producing wildtype. Neither Cal-γ nor α-1-ovoglycoprotein bind salmochelin or enterobactin, nor do they inhibit iron-restricted growth of S. Enteritidis. However, both are present in EW at significant concentrations (5.6 and 233 μM, respectively) indicating that α-1-ovoglycoprotein is the 4th most abundant protein in EW, with Cal-γ and Ex-FABP at 11th and 12th most abundant. Further, we confirm the preference (16-fold) of Ex-FABP for the ferrated form (K d of 5.3 nM) of enterobactin over the iron-free form (K d of 86.2 nM), and its lack of affinity for salmochelin. In conclusion, our findings show that salmochelin production by S. Enteritidis enables this key egg-associated pathogen to overcome the enterobactin-sequestration activity of Ex-FABP when this lipocalin is provided at levels found in EW.

Microbial spoilage of liquid egg products and its control by the detection of genetic or chemical markers. journée scientifique "science des aliments et valorisation des bio-produits"

Conference: 11th International Symposium on Biometals (Biometals) Location: Ottawa, CANADA Date: 2018; Salmonella enterica serovar Enteritidis (SE) is the most frequently-detected Salmonella in foodborne outbreaks in the European Union. Among such outbreaks, egg and egg products were identifiedas the most common vehicles of infection. Possibly, the major antibacterial property of egg white is ironrestriction, which results from the presence of the ironbinding protein, ovotransferrin. To circumvent ironrestriction, SE synthesise catecholate siderophores (i.e. enterobactin and salmochelin) that can chelateiron from host iron-binding proteins. Here, we highlight the role of lipocalin-like proteins found in eggwhite that could enhance egg-white iron restriction through sequestration of certain siderophores, includingenterobactin. Indeed, it is now apparent that the egg-white lipocalin, Ex-FABP, can inhibit bacterialgrowth via its siderophore-binding capacity in vitro. However, it remains unclear whether Ex-FABP performssuch a function in egg white or during bird infection. Regarding the two other lipocalins of eggwhite (Cal-c and a-1-glycoprotein), there is currently no evidence to indicate that they sequestersiderophores.

National audience; Salmonella outbreaks are a serious threat to public health. One of the main vehicles of infection are eggs and egg-products, with Salmonella enterica serovar Enteritidis being the most frequent contaminant. Egg contamination occurs despite the impressive antibacterial properties of egg white and it is notable that S. Enteritidis is able to survive in egg white in contrast to other serovars and species. It is generally accepted that the major egg white-factor limiting growth of Salmonella is the highly iron-restricted environment resulting from the presence of ovotransferrin, an iron-binding protein. To circumvent iron restriction, many bacteria synthesise siderophores that can chelate iron from host iron-binding proteins for delivery to bacteria. The iron starvation response genes are regulated according to iron availability via the transcriptional regulator, Fur. Evidence for a relationship between temperature and iron sequestration ability has been reported by several groups. For Salmonella Typhymurium, little or no siderophore is secreted at 40-42°C in iron depleted medium indicating a temperature regulatory effect that is independent of Fur/Fe control. The aim of this study is to determine if egg white exposure induces the iron-acquisition genes and siderophore production in the highly-egg-white adapted Enteritidis serovar at ~40°C, the natural hen body temperature that Salmonella would encounter when infecting the egg during egg formation in the oviduct or during hatching. In order to easily recover of S. Enteritidis, we used egg white model medium (EWMM) in place of viscous egg white. EWMM consists of egg white filtrate with 10% egg white and numerous studies have validated this model medium. We measured the expression of 15 genes involved in the ironrestriction response by qRT-PCR after S. Enteritidis incubation of 45 minutes in EWMM at 37, 40 and 42°C. We observed a strong expression of genes encoding proteins involved in biosynthesis, export, uptake and utilisation of enterobactin and salmochelin (the two siderophores of Salmonella) and ferric-hydroxamate uptake. A statistical analysis shows that iron-acquisition genes are better expressed at 40°C than at 37 or 42°C. The production of siderophores was quantified during incubation using the Calcein blue test. Siderophores released by Salmonella in EWMM were detected at around a 1 μM desferral equivalent within 6h of incubation at 37, 40 and 42°C. The results of this study suggest that S. Enteritidis is able to induce its iron acquisition systems to cope with the iron-restricted conditions imposed by egg white even at the elevated body temperature of the hen.

International audience; Selon l'Organisation Mondiale de la Santé, 420 000 personnes dont 125 000 enfants meurent chaque année d'infections alimentaires. Pour l'Europe, le nombre de décès s'élève à 260 personnes pour l'année 2014. Les maladies diarrhéiques sont responsables de plus de la moitié des décès. En France, les salmonelles expliquent à peu-près 50% des infections alimentaires. Dans ce contexte, cette communication investit le scénario d'une toxi-infection à la salmonelle de laits infantiles de premier âge. Notre objectif est d'estimer économiquement et humainement l'impact d'une telle crise sur le système sanitaire français. Le modèle de simulation employé est un programme mathématique reproduisant une version étendue du modèle SIR. Il permet l'expérimentation de différentes variantes de notre scénario.

Among the nucleic regulators, small regulatory RNAs are a quick way to adapt. They are found in all areas of life. In bacteria, the action of a large number of them depends on the chaperone protein of Hfq RNA. Their mode of action consists of matching directly to the target mRNA by imperfect complementary sequence. This results in a positive or negative control, translation and/or stability of the target mRNA.During the first part of this thesis, we have tried to characterize the regulation of mRNA by small regulatory RNAs. To do this, we studied the different components of this regulation and we have shown that the binding sites on protein Hfq on RNA should be consistent for optimal regulation of mRNA by small regulatory RNAs.In the second part of the thesis, we identified a new type of mRNA regulation by small RNA regulators: transcriptional polarity. We could show that the fixing of small RNA ChiX on the 5 'UTR region of the mRNA chiPQ cause inhibition of translation of chiP but also a Rho-dependent premature termination of mRNA transcription.We subsequently investigated whether this type of regulation could be in the control of other mRNAs as dppABCDF’s operon and pgaABCD’s operon. In parallel with these studies, we have also tried characterized Rho-dependent termination between S. Typhimurium and E. coli in mRNA chiPQ., Parmi les régulateurs nucléiques, les petits ARN régulateurs sont un moyen rapide d’adaptation. On les retrouve dans tous les domaines du vivant. Chez les bactéries, l’action d’un grand nombre d’entre eux dépend de la protéine chaperonne d’ARN Hfq. Leur mode d’action consiste à d’apparier directement à l’ARNm cible par complémentarité imparfaite de séquence. Il en résulte une régulation positive ou négative, de la traduction et/ou de la stabilité de l’ARNm cible.Au cours de la première partie cette thèse, nous avons essayé de caractériser la régulation des ARNm par les petits ARN régulateurs. Pour ce faire, nous avons étudiés les différents composants de cette régulation et nous avons pu montrer que les sites de fixation à la protéine Hfq sur les ARN devaient être compatibles pour obtenir une régulation optimale des ARNm par les petits ARN régulateurs.Lors de la seconde partie de la thèse, nous avons pu identifier un nouveau type de régulation des ARNm par les petits ARN régulateurs : la polarité transcriptionnelle. Nous avons pu montrer que la fixation du petit ARN ChiX sur la région 5’UTR de l’ARNm chiPQ entrainer l’inhibition de la traduction de chiP mais également une terminaison prématurée rho-dépendante de la transcription de l’ARNm.Nous avons par la suite cherché à savoir si ce type de régulation pouvait se retrouver dans la régulation d’autre ARNm comme l’opéron dppABCDF et l’opéron pgaABCD. En parallèle de ces études, nous avons également essayé de caractérisé la terminaison Rho-dépendante entre S. Typhimurium et E.coli au sein de l’ARNm chiPQ.

Les anevrismes infectieux de l'axe femoro-poplite sont rares et d'evolution silencieuse. Ils surviennent surtout chez les sujets immunodeprimes. Leur traitement fait appel souvent a l'exclusion chirurgicale associee ou non a un geste de revascularisation. Nous rapportons le cas d'un patient opere pour un anevrisme de l'artere femorale superficielle, lie a une infection a salmonelle, il a eu une resection de l'anevrysme et une revascularisation par greffe veineuse. Ce patient a ete repris par la suite a deux reprise pour rupture du greffon veineux. Pour cette raison, nous etions contraints a une explantation du pontage et une ligature des 2 bouts de l'artere sans revascularisation. Malgre l'absence de revascularisation, l'evolution a ete favorable sous couverture d'une une antibiotherapie adaptee. En cas d'anevrysme infectieux de l'axe femoropoplite par infection a salmonelle, le retablissement de la continuite arterielle, meme avec un greffon veineux, peut exposer au risque de rupture arterielle. Key words: Anevrisme mycotique, artere femorale superficielle, salmonelle

Les anévrismes infectieux de l'axe fémoro-poplité sont rares et d’évolution silencieuse. Ils surviennent surtout chez les sujets immunodéprimés. Leur traitement fait appel souvent à l'exclusion chirurgicale associée ou non à un geste de revascularisation. Nous rapportons le cas d'un patient opéré pour un anévrisme de l'artère fémorale superficielle, lié à une infection à salmonelle, il a eu une résection de l'anévrysme et une revascularisation par greffe veineuse. Ce patient a été repris par la suite à deux reprise pour rupture du greffon veineux. Pour cette raison, nous étions contraints à une explantation du pontage et une ligature des 2 bouts de l'artère sans revascularisation. Malgré l'absence de revascularisation, l’évolution a été favorable sous couverture d'une une antibiothérapie adaptée. En cas d'anévrysme infectieux de l'axe fémoro-poplité par infection à salmonelle, le rétablissement de la continuité artérielle, même avec un greffon veineux, peut exposer au risque de rupture artérielle.

Salmonella enterica serovar Enteritidis is the prevalent egg-product-related food-borne pathogen. The egg-contamination capacity of S. Enteritidis includes its exceptional survival capability within the harsh conditions provided by egg white. Egg white proteins, such as lysozyme and ovotransferrin, are well known to play important roles in defence against bacterial invaders. Indeed, several additional minor proteins and peptides have recently been found to play known or potential roles in protection against bacterial contamination. However, although such antibacterial proteins are well studied, little is known about their efficacy under the environmental conditions prevalent in egg white. Thus, the influence of factors such as temperature, alkalinity, nutrient restriction, viscosity and cooperative interactions on the activities of antibacterial proteins in egg white remains unclear. This review critically assesses the available evidence on the antimicrobial components of egg white. In addition, mechanisms employed by S. Enteritidis to resist egg white exposure are also considered along with various genetic studies that have shed light upon egg white resistance systems. We also consider how multiple, antibacterial proteins operate in association with specific environmental factors within egg white to generate a lethal protective cocktail that preserves sterility.

As a 1st step, this study aimed at investigating the microbial quality of liquid egg white in a French eggprocessing company. Thirty raw and 33 pasteurized liquid egg white samples were analyzed. Pasteurization was globally found efficient on mesophilic contaminants (1.7 ± 1.6 and 0.8 ± 0.9 log CFU/mL in raw and pasteurized samples, respectively), including for the control of Salmonella. However,Gram-positive enterococci were still detected in the pasteurized samples. As a 2nd step, a representative bacterial collection was built for exploring the spoilage issue in egg-based chilled desserts. Custard cream was chosen as growth medium since this food is widely used for the production of French chilled desserts. All of the 166 isolates of the bacterial collection were shown to be able to grow and to induce spoilage of the custard cream at refrigeration temperature (10 °C). Several spoilage types were highlighted in the custard cream, on the basis of changes regarding pH, consistency, production of holes or gas. As a 3rd step, bacterial enzymatic activities were explored on custard cream-based agar media. The bacterial collection was reduced to 43 isolates, based on further selection regarding the genera and the spoilage types previously highlighted. Albeit to different degrees, all these isolates were able to produce proteases. A large part of these isolates also expressed lipolytic and amylolytic activities. This studyemphasizes the need to control egg white contamination and especially with Gram-positive heat-resistant Enterococi, in order to guarantee the shelf life of egg-based chilled desserts.

The objective of this study was to develop and evaluate a SYBR Green real time PCR method for the specific detection of Salmonella spp using a novel target, the siiA gene. Primer specificity testing was done on a panel of 76 Salmonella strains and 32 non-Salmonella strains. The primers directed against the siiA gene amplified all Salmonella strains tested, while non-Salmonella strains were not amplified. The melting temperatures of the 107 bp amplicons were consistently specific as they gave melting peaks around 75.5 °C. The precision of the assay, based on intra and inter-run variations, was shown to be widely acceptable. In the second part of this study, 45 Salmonella strains were screened for the presence of 6 virulence-associated genes (sopB, cat2, safC, sefB and SC1248) located in several Salmonella Pathogenicity Islands (SPIs) and the spvC gene from the Salmonella virulence plasmid. The prevalence of these genes ranged from 51% to 100%. Variable virulence gene profiles were obtained even within the same serotype.

Liste des auteurs cf: EFSA Panel on Biological Hazards (BIOHAZ): biohaz@efsa.europa.euEuropean Food Safety Authority (EFSA); Salmonella Enteritidis is considered the only pathogen currently posing a major risk of egg-borne diseases in the European Union (EU). The possible impact of extending the shelf-life of eggs on the risk to consumers posed by S. Enteritidis was estimated by applying a quantitative model and comparing the actual situation regarding the storage of eggs in the EU with different possible scenarios combined, considering the prolongation of the best-before and the sell-by date from 7 to a maximum of 70 days. Extending the sell-by date by one week (from 21 to 28 days), but leaving the best-before date unchanged, is estimated to result in a relative risk of illness of 1.4 and 1.5 for uncooked and lightly cooked egg meals respectively, compared to the current situation. If the best-before date is also extended by one week (from 28 to 35 days), the relative risk would be 1.6 and 1.7. In the worst case scenario considered (sell-by date of 42 days, best before date of 70 days), such figures would be 2.9 and 3.5. It should be noted that the absolute risk is greater for uncooked meals compared to lightly cooked meals. An effective way to minimise any increase in risk during extended storage is to keep the eggs refrigerated both at retail and the household. Regarding egg spoilage, such events strongly depend on the hygienic conditions of egg production and practices of egg handling, including storage times and temperatures. Finally, the impact of the prolongation of storage time on the quality criteria for eggs (3-hydroxybutyric acid and lactic acid) destined for manufacturing of egg products is considered negligible.

All over the world, the incidence of Salmonella spp contamination on different food sources like broilers, clams and cow milk has increased rapidly in recent years. The multifaceted properties of Salomnella serovars allow the microorganism to grow and multiply in various food matrices, even under adverse conditions. Therefore, methods are needed to detect and trace this pathogen along the entire food supply network. In the present work, PFGE and ERIC-PCR were used to subtype 45 Salmonella isolates belonging to different serovars and derived from different food origins. Among these isolates, S. Enteritidis and S. Kentucky were found to be the most predominant serovars. The Discrimination Index obtained by ERIC-PCR (0.85) was slightly below the acceptable confidence value. The best discriminatory ability was observed when PFGE typing method was used alone (DI = 0.94) or combined with ERIC-PCR (DI = 0.93). A wide variety of profiles was observed between the different serovars using PFGE or/and ERIC-PCR. This diversity is particularly important when the sample origins are varied and even within the same sampling origin.

Communication orale - Session I : Maîtriser les risques. Première partie : Comprendre/caractériser et gérer les risques de la matière première aux consommateurs; National audience; Salmonella are the bacterial pathogens responsible for typhoid diseases and gastroenteritis which are of public health concern worldwide. Contaminated water is the main source of contamination in developing countries while raw or undercooked foods, especially those of avian origin, are mainly incriminated in industrialized countries. Recently, contaminated plants have also been involved. Thanks to scientific advances in the understanding of the bacterial/host interactions, in particular the interaction of Salmonella with the animal intestine or with plants, methods can be proposed to fight Salmonella infections. These bacterial/host interactions are complex, involving both bacterial and host factors. Studies that have been performed in poultry have helped to establish prophylactic methods based on hygiene measures, the development of vaccine or competitive flora, or the selection of lines resistant to Salmonella infection or carrier state. All of these approaches contribute to the reduction of the colonisation of poultry by Salmonella in order to reduce human contamination.; Les salmonelles sont des bactéries pathogènes responsables des fièvres typhoïde et paratyphoïde et de gastroentérites qui constituent un problème très important en santé publique dans le monde. Alors que dans les pays en voie de développement, l’eau est la principale source de contamination, dans les pays industrialisés, les aliments consommés crus ou peu cuits, en particulier les produits d’origine aviaire, constituent la source majeure de contamination humaine. Récemment, des végétaux ont également été incriminés. C’est grâce aux avancées réalisées sur la compréhension des mécanismes d’interaction entre les bactéries et leurs hôtes, notamment l’interaction des salmonelles avec les végétaux ou avec l’intestin des animaux que des méthodes peuvent être proposées pour lutter contre les infections à salmonelles. Ces interactions sont complexes et impliquent à la fois des facteurs de la bactérie et des facteurs de l’hôte. Les recherches menées chez les volailles permettent de proposer des méthodes de prophylaxie qui reposent sur la définition de mesures d'hygiène, le développement de vaccins ou de flores intestinales compétitives, mais aussi sur la sélection de lignées de volailles résistantes à l'infection ou au portage des salmonelles. Toutes ces approches visent à réduire la contamination des volailles afin de diminuer la contamination humaine.

Les salmonelles sont des bactéries pathogènes responsables des fièvres typhoïde et paratyphoïde et de gastroentérites qui constituent un problème très important en santé publique dans le monde. Alors que dans les pays en voie de développement, l’eau est la principale source de contamination, dans les pays industrialisés, les aliments consommés crus ou peu cuits, en particulier les produits d’origine aviaire, constituent la source majeure de contamination humaine. Récemment, des végétaux ont également été incriminés. C’est grâce aux avancées réalisées sur la compréhension des mécanismes d’interaction entre les bactéries et leurs hôtes, notamment l’interaction des salmonelles avec les végétaux ou avec l’intestin des animaux que des méthodes peuvent être proposées pour lutter contre les infections à salmonelles. Ces interactions sont complexes et impliquent à la fois des facteurs de la bactérie et des facteurs de l’hôte. Les recherches menées chez les volailles permettent de proposer des méthodes de prophylaxie qui reposent sur la définition de mesures d'hygiène, le développement de vaccins ou de flores intestinales compétitives, mais aussi sur la sélection de lignées de volailles résistantes à l'infection ou au portage des salmonelles. Toutes ces approches visent à réduire la contamination des volailles afin de diminuer la contamination humaine., Salmonella are the bacterial pathogens responsible for typhoid diseases and gastroenteritis which are of public health concern worldwide. Contaminated water is the main source of contamination in developing countries while raw or undercooked foods, especially those of avian origin, are mainly incriminated in industrialized countries. Recently, contaminated plants have also been involved. Thanks to scientific advances in the understanding of the bacterial/host interactions, in particular the interaction of Salmonella with the animal intestine or with plants, methods can be proposed to fight Salmonella infections. These bacterial/host interactions are complex, involving both bacterial and host factors. Studies that have been performed in poultry have helped to establish prophylactic methods based on hygiene measures, the development of vaccine or competitive flora, or the selection of lines resistant to Salmonella infection or carrier state. All of these approaches contribute to the reduction of the colonisation of poultry by Salmonella in order to reduce human contamination.

International audience

chapitre 8; Qualité microbiologique de l'oeuf coquille

The IL-12-IFN-γ axis plays an important role in the immunity against mycobacteria. I have identified and studied a cohort of patients with a complete autosomal recessive IL12RB1 deficiency coding for the β1 subunit of the IL-12 and IL-23 receptors. We herein report an international survey of 137 patients from 101 kindreds and 30 countries. A total of 52 IL12RB1 mutant alleles were found. All patients had a functional complete IL-12Rβ1 deficiency, most with a lack of IL-12Rβ1 expression at the cell surface. Clinical phenotypes are heterogeneous from an absence of infection to the death following infection. In most cases, infection consisted in mycobacterial diseases (BCG, environmental mycobacteria and tuberculosis) and/or salmonella diseases. Candidiasis was also being frequently associated to this defect. The IL-23-IL-17 axis seems to play a role in the differentiation and activation of the Th17 CD4+ T cells. The cytokines controlling the development of these cells are not well known. We addressed the question of the development of human IL-17-producing T helper cells in vivo by quantifying the production and secretion of IL-17 by fresh T cells ex vivo, and by T cell blasts expanded in vitro from patients with particular genetic disorders affecting TGF-β (patients TGFB1, TGFBR1 and TGFBR2), IL-1β (patients IRAK4 and MYD88), IL-6 (patients STAT3), or IL-23 (patients IL12B and IL12RB1) responses. Mutations in STAT3 and, to a lesser extent mutations in IL12B and IL12RB1, impaired the development of IL-17- producing T cells. These data suggest that these molecules play a key role in the differentiation and/or expansion of human IL-17-producing T cell populations in vivo, L'axe IL-12-IFN-γ joue un rôle important dans l'immunité anti-mycobactérienne. J'ai identifié et étudié une cohorte de 137 patients présentant un déficit autosomique récessif complet d'IL12RB1 qui code la sous-unité β1 des récepteurs de l'IL-12 et de l'IL-23. Ces patients sont issus de 101 familles provenant de 30 pays. Ils présentent une grande diversité génétique avec 52 allèles mutants différents. Le phénotype cellulaire avec un défaut complet de réponse à l'IL-12 est homogène chez tous les patients. Les phénotypes cliniques sont eux très hétérogènes allant de l'absence d'infection jusqu'au décès. Il s'agit en grande majorité d'infections mycobactériennes (BCG, mycobactéries environnementales et tuberculose) et/ou à salmonelles. La candidose est aussi retrouvée associée à ce défaut chez un grand nombre de patients. L'axe IL-23-IL-17 participe à la différentiation et à l'activation des lymphocytes T CD4+ dits de type Th17. les cytokines et les mécanismes contrôlant la différentiation de ces cellules sont peu connus. J'ai étudié le développement des lymphocytes producteurs d'IL-17 chez des patients porteurs de défauts génétiques affectant la voie du TGF-β (patients TGFBR1, TGFBR2 et TGFB1), de l'IL-1β (patients IRAK4 et MYD88), de l'IL-6 (patients STAT3) et de l'IL-23 (patients IL12B et IL12RB1). Pour cela, j'ai quantifié la production et la sécrétion d'IL-17 dans deux modèles expérimentaux ex vivo et in vitro. Les patients IL12B-/- et IL12RB1-/-, et de façon plus drastique les patients STAT3-/- présentent une diminution des lymphocytes producteurs d'IL-17, ce qui suggère l'importance de ces molécules dans la différentiation et l'expansion des cellules Th17 in vivo

Des QTL aux gènes de résistance au portage de salmonelles chez le poulet

Increasing resistance to acute Salmonellosis (that is, contamination level shortly after infection) is not sufficient to reduce the risk for consumers to be contaminated by Salmonella. Indeed, animals may remain contaminated at a low level for weeks or months. Increased resistance to the Salmonella carrier state, i.e., animals' ability to clear bacteria, is needed; it involves measuring bacterial contamination several weeks after inoculation with a low dose. To study such resistance traits, three convergent approaches were used. A quantitative trait loci (QTL) study was performed, taking advantage of inbred lines differing in resistance. Several QTLs controlling resistance at a younger age were identified and are currently being confirmed in a new cross before finer mapping, using advanced intercross lines. These inbred lines are also presently being compared using functional genomics. In parallel, a selection experiment for increased or decreased resistance at a younger and a later age was undertaken. Besides providing genetic models differing in their levels of resistance, it underlined the importance of the choice of selection criterion, whether marker assisted or not. Indeed, genes controlling resistance are strongly dependant on age; selecting for resistance at a younger age might result in increased susceptibility at an older age. Finally, the results of this experiment were used in a model of the intra-flock propagation of Salmonella. It showed that introducing a proportion of resistant animals within a flock of susceptible hens could dramatically change the evolution of contamination. Moreover, it demonstrated the magnitude of synergy between selection and vaccination, which should enhance the interest of increased resistance. The results show that selection for increased resistance to the Salmonella carrier state may be efficient, providing that the appropriate criteria of selection are used.

The IL-12-IFN-γ axis plays an important role in the immunity against mycobacteria. I have identified and studied a cohort of patients with a complete autosomal recessive IL12RB1 deficiency coding for the β1 subunit of the IL-12 and IL-23 receptors. We herein report an international survey of 137 patients from 101 kindreds and 30 countries. A total of 52 IL12RB1 mutant alleles were found. All patients had a functional complete IL-12Rβ1 deficiency, most with a lack of IL-12Rβ1 expression at the cell surface. Clinical phenotypes are heterogeneous from an absence of infection to the death following infection. In most cases, infection consisted in mycobacterial diseases (BCG, environmental mycobacteria and tuberculosis) and/or salmonella diseases. Candidiasis was also being frequently associated to this defect. The IL-23-IL-17 axis seems to play a role in the differentiation and activation of the Th17 CD4+ T cells. The cytokines controlling the development of these cells are not well known. We addressed the question of the development of human IL-17-producing T helper cells in vivo by quantifying the production and secretion of IL-17 by fresh T cells ex vivo, and by T cell blasts expanded in vitro from patients with particular genetic disorders affecting TGF-β (patients TGFB1, TGFBR1 and TGFBR2), IL-1β (patients IRAK4 and MYD88), IL-6 (patients STAT3), or IL-23 (patients IL12B and IL12RB1) responses. Mutations in STAT3 and, to a lesser extent mutations in IL12B and IL12RB1, impaired the development of IL-17- producing T cells. These data suggest that these molecules play a key role in the differentiation and/or expansion of human IL-17-producing T cell populations in vivo, L'axe IL-12-IFN-γ joue un rôle important dans l'immunité anti-mycobactérienne. J'ai identifié et étudié une cohorte de 137 patients présentant un déficit autosomique récessif complet d'IL12RB1 qui code la sous-unité β1 des récepteurs de l'IL-12 et de l'IL-23. Ces patients sont issus de 101 familles provenant de 30 pays. Ils présentent une grande diversité génétique avec 52 allèles mutants différents. Le phénotype cellulaire avec un défaut complet de réponse à l'IL-12 est homogène chez tous les patients. Les phénotypes cliniques sont eux très hétérogènes allant de l'absence d'infection jusqu'au décès. Il s'agit en grande majorité d'infections mycobactériennes (BCG, mycobactéries environnementales et tuberculose) et/ou à salmonelles. La candidose est aussi retrouvée associée à ce défaut chez un grand nombre de patients. L'axe IL-23-IL-17 participe à la différentiation et à l'activation des lymphocytes T CD4+ dits de type Th17. les cytokines et les mécanismes contrôlant la différentiation de ces cellules sont peu connus. J'ai étudié le développement des lymphocytes producteurs d'IL-17 chez des patients porteurs de défauts génétiques affectant la voie du TGF-β (patients TGFBR1, TGFBR2 et TGFB1), de l'IL-1β (patients IRAK4 et MYD88), de l'IL-6 (patients STAT3) et de l'IL-23 (patients IL12B et IL12RB1). Pour cela, j'ai quantifié la production et la sécrétion d'IL-17 dans deux modèles expérimentaux ex vivo et in vitro. Les patients IL12B-/- et IL12RB1-/-, et de façon plus drastique les patients STAT3-/- présentent une diminution des lymphocytes producteurs d'IL-17, ce qui suggère l'importance de ces molécules dans la différentiation et l'expansion des cellules Th17 in vivo

In Deutschland wird die Salmonellenprävalenz in Schweinebeständen derzeit serologisch mithilfe drei verschiedener kommerzieller, auf Salmonellen-Lipopolysaccharid (LPS) basierender Tests ermittelt. Nachteilig ist hier, dass diese Tests hauptsächlich Antikörper der Immunglobulinklasse G (IgG) erkennen. Ein neuartiger Test erkennt dagegen gleichzeitig drei Ig-Klassen (IgM, IgG und IgA). Anhand verschiedener Konstellationen dieser drei Ig-Klassen zueinander ist es möglich, eine Aussage über den Infektions- oder Impfstatus eines einzelnen Tieres zu treffen. Dies wurde anhand drei verschiedener, von einander unabhängiger Impfmodellversuche mit einem zugelassenen Salmonella-Typhimurium-Lebendimpfstoff unter Feldbedingungen belegt.

Ce travail expérimental présente une caractérisation clinique et bactériologique de l'infection mammaire salmonellique chez la brebis. Après avoir effectué une étude bibliographique des infections salmonelliques chez les bovins et les ovins, l'auteur présente les résultats du suivi de huit brebis spontanément excrétrices de salmonelles dans le lait. Ces dernières excrètent majoritairement une salmonelle du sous-groupe III, Salmonella arizonae, de façon quantitativement irrégulière. Cette infection provoque le plus souvent des mammites subcliniques avec hypogalactie et élévation des valeurs des comptages de cellules somatiques du côté excréteur. Salmonella arizonae reconnaît ainsi un portage mammaire, mais également digestif. Aucun symptôme extra-mammaire n'a cependant été mis en évidence. Ce travail a permis de mieux caractériser l'une des sources majeures de contamination salmonellique des laits et de renforcer en conséquence le plan de maîtrise du risque spécifique chez les ovins.

Effets des fumonisines sur la santé du porc : sensibilité aux salmonelles et statut immunitaire. 38. Journées de la Recherche Porcine

Performance indicators are variables used to calculate on a continuous basis the operational level of a surveillance network's priority activities. A 10-step process was developed to enable network coordinators to identify specific performance indicators to help them monitor their networks. The methodology was based on a listing of surveillance activities, the definition and choice of network's global priority objectives, the construction of performance indicators and monitoring tables and the implementation of the system within the network. This process was implemented for the bovine clinical digestive salmonellosis surveillance network in France (RESSAB). The process produced a list of 26 activities synthesized into 15 global objectives, from which 12 were retained as priority objectives. This made it possible to develop 12 performance indicators. Indicators retrospectively calculated for the year 2003 indicated that RESSAB was operating according to the objectives set by the network's stakeholders and its financial supporter. The methodology was implemented successfully and was not very time consuming (12 person-days) or expensive. The decision makers and actors in the network quickly appropriated the system. The methodology can thus be considered validated through this example. Nevertheless, the risks inherent in the use of performance indicators must be addressed by ensuring the relevance of the selected indicators through external assessment and by prioritizing an internal and participatory approach to avoid a misuse of the performance indicators. In addition, considering that performance indicators address only the operation of the network, relevance of surveillance procedure should be addressed by external evaluation.

Diplôme : Dr. d'Université; La persistance asymptomatique de Salmonella Enteritidis au niveau du tube digestif des volailles est une préoccupation majeure pour la sécurité alimentaire. Les mécanismes de ce portage sont encore mal connus. L'objectif de la thèse était de mettre en évidence les facteurs de l'immunité innée intestinale qui pouvaient être déficients chez les oiseaux fortement porteurs. Pour cela nous avons analysé l'expression différentielle des gènes de médiateurs inflammatoires et anti-infectieux en comparant des lignées de volaille plus ou moins porteuses de salmonelles. Nous avons pu montrer en particulier que la résistance au portage variait selon l'âge des animaux et que l'expression élevée des gènes de cytokines inflammatoires et des défensines était liée à un meilleur contrôle de la colonisation chez l'adulte mais pas chez le jeune. Ces résultats suggèrent que l'efficacité de ces facteurs dans la lutte des oiseaux contre le portage dépend de la maturation du système immunitaire.

RESSAB is a clinical epidemiosurveillance network for salmonellosis diarrhoea in adult cattle. Since its creation at the end of 1996, the network has collected a large amount of data on the clinical features of the disease, its extension in affected herds, the contamination of milk or liquid manure in dairy farms, and the contamination of suckling beef calves, or on serovar distribution and antimicrobial resistance. The annual incidence of this disease is approximately one herd per thousand and seems to be stable since 2000. However this disease should not be underestimated because its clinical features are not specific enough to make a positive diagnosis and because it has a major impact on public health., Le RESSAB, réseau d’épidémiosurveillance clinique des diarrhées à salmonelles des bovins adultes a permis, depuis sa création fin 1996, de collecter de nombreuses informations sur la clinique des animaux atteints, l’extension de la maladie dans les troupeaux concernés, la contamination des laits ou du lisier en élevage laitier et celle des veaux en élevage allaitant ou encore sur la distribution des sérovars et la résistance acquise aux antibiotiques. L’incidence annuelle de cette maladie est d’environ un troupeau pour 1000 et apparaît être stable depuis 2000. Cette maladie ne doit pas être sous-estimée d’autant que le tableau clinique ne permet pas de faire un diagnostic de certitude et que les conséquences hygiéniques sont importantes., Chazel Myriam, Buret Yves, Meunier Danièle, Calavas Didier. Le RESSAB, réseau d'épidémiosurveillance des salmonelloses bovines : fonctionnement et résultats. In: Bulletin de l'Académie Vétérinaire de France tome 158 n°4, 2005. pp. 347-351.

L'auteur présente une synthèse bibliographique sur les Salmonelles et les flores de barrière. Puis, elle présente les résultats d'une étude visant à apprécier l'efficacité d'une flore de barrière indéfinie contre les contaminations salmonelliques chez le poulet label dans le Sud-Ouest. 69 élevages appartenant à 4 organisations de production ont été inclus dans l'étude sur des critères de positivité en Salmonelles à partir de contrôles de fin de lot et ont été répartis en 2 groupes. les élevages d'un groupe reçoivent des poulets à qui on a administré de la flore de barrière tandis que les autres servent de témoin. 3.5% des fonds de boîte issus des poulets ayant été traités avec la flore de barrière à un jour sont positifs en Salmonelles et 9.1% des pédichiffonnettes de fin de lot contre, respectivement, 5.13% des fonds de boîte et 6,8% des pédichiffonnettes sur les lots témoins. Les résultats zootechniques des deux groupes ne sont pas significativement différents. Ces résultats, marqués par la faible prévalence des contaminations par les Salmonelles (6,2%), ne permettent pas de conclure quant à l'efficacité de la flore de barrière dans cet essai sur le terrain.

Le portage asymptomatique de Salmonella enterica par les porcs pose un problème de santé publique et constitue des enjeux économique et commercial majeurs. De nombreux pays vont ou ont déjà mis en place des plans de contrôle nationaux sur toute la chaîne de production porcine. Un projet de directive européenne est actuellement en cours de rédaction et de négociation. Actuellement aucune mesure n'a été mise en place en France. Nous nous devons d'investir ce sujet afin d'être prêt à répondre aux futures exigences réglementaires et commerciales. Cette étude, réalisée au sein du groupement de producteurs bretons, Coopagri-Bretagne, présente une approche de la problématique Salmonella de l'élevage à l'abattoir. La séroprévalence en salmonelles a été estimée à partir de 113 élevages à 46%. Un questionnaire a permis de mettre en évidence un facteur de risque d'une contamination en élevage par les salmonelles : l'usage d'aliment sec par opposition à l'usage d'aliment fermenté liquide. Les mesures de nettoyage- désinfection et de protection sanitaire ne semblent pas, selon notre étude, être associées au statut salmonellique des élevages. Pour caractériser le statut d'un élevage de porc, la bande est l'unité de référence et la sérologie semble être le test diagnostique le plus adapté. L'excrétion de salmonelles augmente de l'élevage à l'abattoir. L'étape transport- attente à l'abattoir constitue le principal facteur de risque de la contamination des carcasses.

International audience; Reliable ELISAs were investigated with the aim to select hen lines resistant to Salmonella Enteritidis and producing high levels of antibodies. In the first experiment, the relation between the humoral response and the bacteriological results was assessed on hens from the Y11 resistant line and the L2 susceptible line, orally inoculated with 10$^8$ CFU S. Enteritidis per animal. Anti- lipopolysaccharide (LPS) IgG titres were higher but the liver and spleen were less contaminated in hens from the Y11 line than in hens from the L2 line ($p = 0.013$, 0.031 and 0.026 respectively). In the second experiment, the hens were inoculated orally with $1.7 \times 10^8$ CFU S. Enteritidis per animal in order to select the ELISA methods showing the more significant differences. ELISAs were based on LPS, flagella, LPS from rough (LPS-R) and smooth strains (LPS-S) and detected IgG and IgM antibodies from sera and yolks. No between-line host response variation was observed in the yolk, with LPS-S and R antigens nor with anti-LPS IgM in the sera. Otherwise, significant differences were encountered between hen lines with the ELISAs performed on the sera detecting anti-LPS IgG, anti-flagella IgG or IgM ($p = 0.017$, 0.017 and $p < 0.001$ respectively). When comparing the kinetics of the selected ELISAs, the IgG antibodies against LPS detected between-line variations as early as 1 to 4 weeks pi, whereas with IgG against flagella, the differences were only detected at 1 and 2 weeks pi and with IgM against flagella, the differences were significant at 1, 2, 4 and 8 weeks pi. In conclusion, resistant hen lines producing higher levels of antibodies than the susceptible hen lines may be selected with these ELISAs.; Méthodes ELISA permettant de différencier des lignées de poules résistante et sensible à Salmonella Enteritidis inoculées par voie orale. Des méthodes ELISA ont été recherchées pour sélectionner les lignées de poules résistantes à Salmonella Enteritidis produisant des taux élevés d'anticorps. Dans la première expérience, la relation entre la réponse sérologique et les résultats bactériologiques a été évaluée sur les poules des lignées résistante Y11 et sensible L2, inoculées par voie orale avec 10$^8$ UFC S. Enteritidis par animal. Les titres en anticorps IgG anti- lipopolysaccharidiques (LPS) étaient supérieurs mais les foies et rates étaient moins contaminés chez les poules de la lignée Y11 que chez les poules de la lignée L2 ($p = 0.013$, 0.031 et 0.026 respectivement). Dans la seconde expérience, les deux lignées de poules ont été inoculées par voie orale avec $1.7 \times 10^8$ UFC S. Enteritidis par animal afin de sélectionner les méthodes ELISA les plus discriminantes. Les ELISA utilisaient comme antigènes des LPS, flagelles, LPS des souches rough (LPS-R) et smooth (LPS-S), et détectaient des anticorps IgG et IgM dans le sérum et le vitellus. Aucune différence significative n'a été observée à partir du vitellus, ou avec les antigènes LPS-S et R, ainsi qu'avec les IgM anti-LPS dans le sérum. En revanche, les tests ELISA réalisés sur les sérums et basés sur le dépistage des IgG anti-LPS, des IgG ou IgM anti-flagellaires permettent de montrer une différence significative entre les deux lignées ($p = 0.017$, 0.017 et $p < 0.001$ respectivement). La comparaison des cinétiques obtenues avec les tests ELISAs retenus a montré que les IgG anti-LPS permettaient de détecter des différences entre lignées dès 1 semaine jusqu'à 4 semaines après inoculation, alors qu'avec les IgG anti-flagellaires les différences apparaissaient seulement à 1 et 2 semaines, et avec les IgM anti-flagellaires les différences étaient significatives à 1, 2, 4 et 8 semaines après inoculation. En conclusion, les lignées de poules résistantes et produisant des taux d'anticorps plus élevés que les lignées de poules sensibles peuvent être sélectionnées avec ces tests ELISA.

International audience

International audience

60 to 70 people annually die in Germany by salmonellose but no one by the traces of pesticides or of heavy metals in our food. The author describes the most probable ways of heavy infections of food by microorganisms in the kitchen and how these can be avoided in terms which are understood.

Selection for increased resistance to Salmonella colonisation and excretion could reduce the risk of foodborne Salmonella infection. In order to identify potential loci affecting resistance, differences in resistance were identified between the N and 61 inbred lines and two QTL research performed. In an F2 cross, the animals were inoculated at one week of age with Salmonella enteritidis and cloacal swabs were carried out 4 and 5 wk post inoculation (thereafter called CSW4F2 and CSW4F2) and caecal contamination (CAECF2) was assessed 1 week later. The animals from the (N × 61) × N backcross were inoculated at six weeks of age with Salmonella typhimurium and cloacal swabs were studied from wk 1 to 4 (thereafter called CSW1BC to CSW4BC). A total of 33 F2 and 46 backcross progeny were selectively genotyped for 103 and 135 microsatellite markers respectively. The analysis used least-squares-based and non-parametric interval mapping. Two genome-wise significant QTL were observed on Chromosome 1 for CSW2BC and on Chromosome 2 for CSW4F2, and four suggestive QTL for CSW5F2 on Chromosome 2, for CSW5F2 and CSW2BC on chromosome 5 and for CAECF2 on chromosome 16. These results suggest new regions of interest and the putative role of SAL1.

National audience

373 thirteen-week-old chicks issued from a commercial cross and 312 chickens from the L2 line were intravenously inoculated with 106 Salmonella enteritidis and the numbers of Salmonella in the spleen, liver and genital organs were assessed 3 days later. Heritabilities of the number of Salmonella were estimated at 0.02 ± 0.04 and 0.05 ± 0.05 in the liver; at 0.29 ± 0.07 and 0.10 ± 0.06 in the spleen; and at 0.16 ± 0.05 and 0.11 ± 0.08 in the genital organs, in the first and second experiments, respectively. The difference between the two experiments could result from sampling variations and from differences in the genetic structure of the two populations possibly including both heterosis and additive effects as well as their interaction in the first experiment. Genetic correlations between the number of bacteria in the genital organs and liver (0.56 ± 0.58 and 0.76 ± 0.32 in the first and second experiments, respectively) and spleen (0.37 ± 0.24 and 0.79 ± 0.23) were positive. Moreover a significant within-sire effect of VIL1, a marker gene for NRAMP1, was observed in 117 progeny resulting from 25 informative matings. These results indicate that there are genetic differences in the resistance to visceral infection by S. enteritidis in these commercial egg-laying flocks, and suggest that these differences are at least partly due to genetic polymorphism in the NRAMP1 region.

Chantier qualité GA; International audience; UNLABELLED: ABSTRACT: BACKGROUND: Chicken meat and eggs can be a source of human zoonotic pathogens, especially Salmonella species. These food items contain a potential hazard for humans. Chickens lines differ in susceptibility for Salmonella and can harbor Salmonella pathogens without showing clinical signs of illness. Many investigations including genomic studies have examined the mechanisms how chickens react to infection. Apart from the innate immune response, many physiological mechanisms and pathways are reported to be involved in the chicken host response to Salmonella infection. The objective of this study was to perform a meta-analysis of diverse experiments to identify general and host specific mechanisms to the Salmonella challenge. RESULTS: Diverse chicken lines differing in susceptibility to Salmonella infection were challenged with different Salmonella serovars at several time points. Various tissues were sampled at different time points post-infection, and resulting host transcriptional differences investigated using different microarray platforms. The meta-analysis was performed with the R-package metaMA to create lists of differentially regulated genes. These gene lists showed many similarities for different chicken breeds and tissues, and also for different Salmonella serovars measured at different times post infection. Functional biological analysis of these differentially expressed gene lists revealed several common mechanisms for the chicken host response to Salmonella infection. The meta-analysis-specific genes (i.e. genes found differentially expressed only in the meta-analysis) confirmed and expanded the biological functional mechanisms. CONCLUSIONS: The meta-analysis combination of heterogeneous expression profiling data provided useful insights into the common metabolic pathways and functions of different chicken lines infected with different Salmonella serovars.