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Botulism in wild birds is a widespread and potentially lethal disease raising major conservation issues. Botulism is also of public health concern. Due to the action of botulinum neurotoxins, mostly produced by Clostridium botulinum, botulism can affect wild birds, livestock, and humans. This study is part of a project aimed at improving our understanding of the pathogenesis of botulism in wild avifauna, which is still poorly understood. Indeed, the prevalence and dynamics of C. botulinum in the digestive tract or in bird tissue, whether as intermittent carriage related to environmental contamination or as part of the normal avian microbiota, is still unknown. In this study, we specifically addressed the presence of a healthy carrier status of wild birds, and its role in outbreaks. To answer this question, we monitored the estimated prevalence of C. botulinum in wild birds through samples from banded and swabbed birds as well as from hunted bird organs. Our results do not support the hypothesis of a healthy carriage outside of outbreaks, which raises the question of the bioavailability of the bacterium and toxin in the environment. Finally, the gene encoding botulinum neurotoxin type E was detected in keel muscle from a hunted bird, showing that recommendations on the consumption of wild bird meat are needed following a botulism outbreak.

We are grateful to the ANSES expert committee panel in charge of assessing animal health and animal welfare risk, and to the working group “Botulism,” including the authors as well as Catherine Belloc, Stéphane Bertagnoli, Alain Boissy, Henri-Jean Boulouis, Eric Collin, Jean-Claude Desfontis, David Fretin, Emmanuelle Gilot-Fromont, Etienne Giraud, Nadia Haddad, Viviane Hénaux, Elsa Jourdain, Sophie Le Poder-Alcon, Monique L’Hostis, Gilles Meyer, Elodie Monchatre-Leroy, Pierre Mormède, Carine Paraud, Ariane Payne, Carole Peroz-Sapede, Claire Ponsart, Claude Saegerman, Gaëlle Simon, Michèle Tremblay, Nathalie Arnich, Isabelle Attig, Carole Catastini, Charlotte Dunoyer, Nabila Haddache, and Elissa Khamisse.; International audience; Clostridium botulinum is the main causative agent of botulism, a neurological disease encountered in humans as well as animals. Nine types of botulinum neurotoxins (BoNTs) have been described so far. Amongst these “toxinotypes,” the A, the B and E are the most frequently encountered in humans while the C, D, C/D and D/C are mostly affecting domestic and wild birds as well as cattle. In France for instance, many cases and outbreaks are reported in these animal species every year. However, underestimation is very likely at least for avifauna species where the detection of dead animals can be challenging. Knowledge about BoNTs C, D, C/D, and D/C and the diseases they cause in animals and humans is still scarce and unclear. Specifically, the potential role of animal botulism outbreaks in cattle and poultry as a source of human illness needs to be further assessed. In this narrative review, we present the current knowledge about toxinotypes C, D, C/D, and D/C in cattle and poultry with, amongst various other aspects, their epidemiological cycles. We also discuss the zoonotic potential of these toxinotypes and some possible ways of risk mitigation. An adapted and effective management of botulism outbreaks in livestock also requires a better understanding of these less common and known toxinotypes.

The authors thank Marianne Chemaly, Sandra Rouxel, Typhaine Poezevara, Amandine Avouac, Emmanuelle Houard, from the National Reference Laboratory for Botulism, ANSES Ploufragan-Plouzané-Niort Laboratory and Laure Diancourt, Julie Germond, Jean Sautereau, from the National Reference Centre for Anaerobic Bacteria and Botulism, Institut Pasteur, Paris. The authors are grateful to the ANSES expert committee panel in charge of assessing animal health and animal welfare risk, and to the working group Botulism, including the authors FM, SL, CLM, CM as well as (in alphabetical order): Catherine Belloc, Stéphane Bertagnoli, Alain Boissy, Henri-Jean Boulouis, Frédéric Carlin, Eric Collin, Jean-Claude Desfontis, Michel Federighi, Maria-Eleni Filippitzi, Matthieu Fournier, Philippe Fravalo, David Fretin, Jean-Pierre Ganiere, Emmanuelle Gilot-Fromont, Etienne Giraud, Lionel Grisot, Nadia Haddad, Viviane Hénaux, Didier Hilaire, Elsa Jourdain, Sophie Le Poder-Alcon, Monique L'Hostis, Gilles Meyer, Elodie Monchatre-Leroy, Hervé Morvan, Pierre Mormède, Carine Paraud, Ariane Payne, Carole Peroz-Sapede, Claire Ponsart, Claude Saegerman, Gaëlle Simon, Michèle Tremblay, Cédric Woudstra, Jean-Pierre Vaillancourt.; International audience; Botulism is a human and animal neurological disease caused by the action of bacterial neurotoxins (botulinum toxins) produced by bacteria from the genus Clostridium . This disease induces flaccid paralysis that can result in respiratory paralysis and heart failure. Due to its serious potential impact on public health, botulism is a closely monitored notifiable disease in France through a case-based passive surveillance system. In humans, this disease is rare, with an average of 10 outbreaks reported each year, mainly due to the consumption of contaminated foods. Type B and to a lesser extend type A are responsible for the majority of cases of foodborne botulism. Each year, an average of 30 outbreaks are recorded on poultry farms, about 20 cases in wild birds and about 10 outbreaks in cattle, involving a large number of animals. Mosaic forms C/D and D/C in birds and cattle, respectively, are the predominant types in animals in France. Types C and D have also been observed to a lesser extent in animals. With the exception of botulinum toxin E, which was exceptionally detected throughout the period in wild birds, the types of botulism found in animal outbreaks are different from those identified in human outbreaks over the last ten years in France and no human botulism outbreaks investigated have been linked to animal botulism. In line with the One Health concept, we present the first integrative approach to the routine surveillance of botulism in humans and animals in France.

International audience; This study examined the combined effect of hydraulic retention time (HRT), organic loading rate (OLR) and heat pretreatment of manure (70 degrees C, 1 h) on the fate of E. coli, enterococci, C. perfringens, C. difficile, and on chemical parameters (volatile fatty acids and ammonia) that may inactivate pathogens. Semi-continuous mesophilic anaerobic reactors were fed with pig manure and horse feed. The operating conditions were 2, 3, 4 COD.L-1.d(-1) (OLR), 24, 35, 46 days (HRT) and use or not of a thermal pretreatment. The levels of the chemical parameters did not reach concentrations capable of inactivating the four bacteria. Anaerobic digestion led to a Log io removal > 3 (E. coli), 0.9-2.1 (enterococci), 0.1-0.6 (C. perfringens) and 0-1 (C. difficile). Increasing HRT only reduced the concentration of E. coli in the digestate. Increasing OLR reduced the Log(10) removal of enterococci and C. difficile. The heat pretreatment led to non-detection of E. coli in the digestate, reduced the concentration of C. perfringens by 0.8-1.3 Log(10) and increased the concentration of C. difficile by 0.04-0.7 Log(10). Enterococci, not detected in the heated manure, were present in the digestate. The distribution of genes encoding virulence factors of C. difficile (tcdA and tcdB) and C. perfringens (cpa, cpb2 and cpb) was not impacted by anaerobic digestion or by the heat pretreatment. Enterococci, C. perfringens, C. difficile were present in the digestate at relatively stable concentrations regardless of the operating conditions, indicating that even with heat pretreatment, the biosafety of digestate cannot be guaranteed in mesophilic conditions.

Digestate produced by agricultural biogas plants (BGPs) may contain pathogenic bacteria. Among them, Clostridium perfringens deserves particular attention due to its ability to grow under anaerobic conditions and persist in amended soil. The aim of this study was to examine the potential pathogenicity and the antimicrobial resistance of C. perfringens in manure and digestate collected from three agricultural biogas plants (BGPs). A total of 157 isolates (92 from manure, 65 from digestate) were screened for genes encoding seven toxins (cpa, cpb, etx, iapcpe, netB, and cpb2). The 138 cpa positive isolates were then screened for tetA(P), tetB(P), tet(M), and erm(Q) genes and tested for antimicrobial susceptibility. The toxinotypes identified in both manure and digestate were type A (78.3% of the isolates), type G (16.7%), type C (3.6%), and type D (1.4%), whereas none of the isolates were type F. Moreover, half of the isolates carried the cpb2 gene. The overall prevalence of tetA(P) gene alone, tetA(P)-tetB(P) genes, and erm(Q) gene was 31.9, 34.8, and 6.5%, respectively. None of the isolates harbored the tet(M) gene. Multiple antimicrobial resistant isolates were found in samples that were collected from all the manure and digestates. Among them, 12.3% were highly resistant to some of the antibiotics tested, especially to clindamycin (MIC &ge, 16 &micro, g/mL) and tilmicosin (MIC &gt, 64 &micro, g/mL). Some isolates were highly resistant to antibiotics used in human medicine, including vancomycin (MIC &gt, 8 &micro, g/mL) and imipenem (MIC &gt, g/mL). These results suggest that digestate may be a carrier of the virulent and multidrug resistant C. perfringens.

A workshop on the risks associated with animal botulism was held in Maisons-Alfort, France in March 2019. The objectives were to debate the public and animal health risks related to botulism, to disseminate the results obtained from the ANIBOTNET project to other European research teams that were not involved in the project and to other stakeholders, and finally to strengthen and broaden the existing research network on this topic. The workshop was divided into 4 sessions: public and animal health risks associated with botulism, a specific focus on risks associated with agricultural biogas plants, dissemination of ANIBOTNET results, and network development and future perspectives. In all, 58 delegates from 13 countries attended the workshop. A summary of the main topics and discussions during the workshop is presented here.

Propionibacterium freudenreichii is a beneficial bacterium used as vitamin producer, bio-preservative, cheese ripening starter and probiotic. It adheres to intestinal epithelial cells and mucus and modulates the immune response. Interaction with the host is strain-dependent and relies on key surface proteins, still poorly investigated. We identified surface-exposed proteins in a strain previously selected for its probiotic properties. The role of these proteins in immunomodulatory properties was evidenced. Cheese being a major source of bacteria, we developed an experimental pressed cheese, exclusively fermented by a single strain of P. freudenreichii. Key surface proteins involved in the observed immunomodulatory effects were expressed within the cheese matrix. Consumption of this single-strain cheese protected mice from acute colitis induced by TNBS. P. freudenreichii expressed the key genes of stress tolerance response during growth in the aqueous phase of Emmental cheese, in agreement with enhanced stress tolerance, compared to laboratory culture medium.This work confirms the probiotic potential of P. freudenreichii, it provides a new functional fermented product for preclinical and clinical studies aimed at prevention or treatment of IBD. It constitutes a basis for further studies aimed at the elucidation of mechanisms responsible for its probiotic effects, in a post-genomic context.

Propionibacterium freudenreichii is a food grade bacterium consumed both in cheeses and in probiotic preparations. Its promising immunomodulatory potential largely relies on the presence of specific and strain-dependent surface compounds. The CIRM BIA 129 strain was selected for immunomodulatory properties, its surfaceome deciphered using an integrative approach. Surface Layer Associated Proteins (SLAPs) are involved in the anti-inflammatory properties

Propionibacterium freudenreichii is the main Propionibacterium consumed both in traditional cheeses and in innovative probiotic product. A portion of 10 grams of Emmental cheese provides around 10 10 live P. freudenreichii , a major bacterial intake in France and other industrialized countries. Surprisingly, little is known about its effect on human health, compared to lactobacilli or bifidobacteria. However, recent works indicate promising beneficial effects including modulation of intestinal microbiota, metabolic activities and inflammation. In this context, the molecular mechanisms responsible for the immunomodulatory properties of P.freudenreichii , and of fermented dairy product containing it, were investigated. In that aim, an integrative approach was undertaken, in a research program of the French National Agency for Research (ANR). It consisted of the sequencing and annotation of the genomes of 20 strains, followed by comparative genomics, in the design of a pangenomic chip, followed by comparative transcriptomic analysis , in a comparative surface proteomic analysis, and in comparison of the immunomodulatory properties, ex vivo and in vivo , using model cheeses. This investigation reveals, for the first time, the molecular mechanism responsible for modulation of the immune system and of inflammation by P.freudenreichii . These results are in accordance with clinical results obtained by others with the same bacterium

Propionibacterium freudenreichii is consumed in elevated amounts both as a cheese starter (1010 CFU/D/person in France) and as a probiotic bacterium, while its effect on health is poorly understood. We investigate its immunomodulatory properties, in a context of growing occurrence of inflammatory bowel diseases. Twenty strains were compared in this regard in vitro using human PBMCs (Peripheral Blood Mononuclear cells), revealing strain dependent cytokine stimulatory properties. They also afforded strain-dependent protection in vivo towards colitis. A fermented milk, exclusively fermented by P. freudenreichii, was developed and tested in a healthy piglet model. It promoted growth health of the piglets and delivered live and metabolically active propionibacteria to the gut. Its consumption reduced gut mucosa production of pro-inflammatory IL-8 and TNF-α. Among strains, a genomic, transcriptomic and proteomic approach revealed contrasted effects of propionibacterial surface compounds such as exopolysaccharides and proteins. Specific proteins are involved in immunomodulation, while capsular exopolysaccharides abolish it. The role of each key compound is presently investigated in an ANR project (Sarter SURFace against INflammation of the Gut) in a multidisciplinary approach involving 7 partners. Elucidation of molecular and cellular mechanisms, together with in vivo anti-inflammatory effects, will open new perspectives for the use of fermented dairy products containing P. freudenreichii addressing the needs of specific populations.

Surface proteins are key actors of the complex interactions between bacteria (pathogens, commensals or symbionts) and their host. In beneficial (probiotic) bacteria, they participate in competition with pathogens, adhesion to the host cells, and immunomodulation. We investigated such proteins in the beneficial bacterium Propionibacterium freudenreichii, consumed both in Swiss-type cheeses and probiotic preparations. P. freudenreichii genome was sequenced and annotated, the localization of the encoded proteins was predicted using SurfG+. A combination of 3 biochemical methods confirmed surface exposure of P. freudenreichii proteins: shedding, shaving and labelling. Shedding consisted in the extraction of cell-wall associated proteins using guanidine, followed by trypsinolysis of the extracted proteins. Shaving consisted in enzymatic hydrolysis of surface protruding proteins which were accessible to trypsin in situ on live bacteria. For labeling, an NHS-ester-cyanine was added to live bacteria in order to label surface proteins, prior to 2-D electrophoresis and detection of fluorescent protein spots. For the 3 methods, the resulting tryptic peptides were identified by NanoLC-MS/MS on a Q-TOF mass spectrometer This combination of methods allowed identification of surface layer type-proteins, lipoproteins, proteins associated to the cell wall, to the membrane, or predicted to be secreted, as well as moonlighting proteins predicted to be cytoplasmic. Some of these proteins are known to participate in adhesion and in the modulation of the immune response by probiotics. This work constitutes a decisive step in the elucidation of P. freudenreichii ability to interact with host cells and in the understanding of protein sorting in this bacterium.

Staphylococcus aureus is an opportunistic pathogen in dairy ruminants where it is a major cause of mastitis and can be also found in healthy carriage. A better knowledge of the host-pathogen interactions is needed to tackle this serious animal health problem. This study aimed at identifying S. aureus proteins differentially expressed during nasal colonization versus mastitis. Serological proteome analysis (SERPA) was used to examine protein samples prepared from culture supernatants of S. aureus strains originally isolated from gangrenous mastitis and nasal carriage (O11) or subclinical mastitis (O46) and to compare patterns of immune-reactive proteins. These staphylococcal proteins were revealed by sera obtained from ewes suffering from S. aureus mastitis and by sera obtained from healthy nulliparous ewes (i.e. no lactation and no mastitis or other symptoms) that were nasally colonized by S. aureus. Altogether 49 staphylococcal immune-reactive proteins were identified in this study. Patterns of proteins revealed by sera from infected- or healthy carrier- animals were comparable and analysis singled out one immune-reactive protein, an N-acetylmuramyl-L-alanine amidase, which was recognized by each of the 6 sera from infected animals, when tested individually, and not by the sera of healthy carriers. This is the first study that compares the S. aureus seroproteome in colonization versus mastitis context in ruminants. These results open avenues for studies aiming at a better understanding of the balance between infection and commensal lifestyle in this opportunistic pathogen and at new prevention strategies.

Dairy propionibacteria, mainly Propionibacterium freudenreichii, are consumed in high amounts within cheeses. They are also known as probiotics able to modulate the gut microbiota and the corresponding metabolic activities. Their immunomodulatory properties, analyzed here on human PBMCs, displayed a remarkable variability, depending on the P. freudenreichii strain. Moreover, such modulating properties varied depending on the culture medium considered, i.e. laboratory culture medium versus fermented dairy product. Selected anti-inflammatory strains were further tested in 2 distinct mice experimental colitis models, revealing significant protection and confirming for these cheese starter bacteria (i) probiotic potential and (ii) predictive value of immune-based selection criteria. The role of surface component, including surface exopolysaccharide (EPS) and surface exposed proteins (SEPs), in this modulation, was investigated. We showed that presence of EPS abrogates immunomodulation while presence of key SEPs determines nature and intensity of this modulation. These key SEPs are being identified, as well as the mechanisms leading to their variability. These results open new perspectives for propionibacteria selection aiming at probiotic applications and new mechanistic explanation of their effects on the digestive tract.

Propionibacterium freudenreichii est une bactérie de l’ordre des Actinomycetales qui bénéficie des statuts GRAS et QPS. Consommée en grande quantité (1010 CFU/jour/personne en France) dans les fromages de type Emmental, elle est également commercialisée en tant que probiotique. Cette étude vise à mettre en évidence les propriétés immunomodulatrices d’une telle consommation, dans un contexte où les maladies intestinales inflammatoires constituent un problème de santé publique croissant. Sujets / matériels et méthodes : Les propriétés immunomodulatrices de 20 souches de P. freudenreichii ont été comparées in vitro dans un modèle de PBMC humains, puis in vivo dans deux modèles de colite expérimentale chez la souris. Un lait fermenté exclusivement par P. freudenreichii a été mis au point et testé dans un modèle de porcelet sain. Une approche multidisciplinaire (purification, mutation, expression hétérologue, évaluation in vitro et in vivo) précise les mécanismes mis en jeux. Résultats principaux : Nous avons mis en évidence des propriétés immunomodulatrices souche-dépendantes pour cette bactérie, en terme de stimulation de cytokines et de protection vis-à-vis de colite expérimentale chez la souris. Le lait fermenté mis au point a eu un effet promoteur de santé chez le porcelet et délivre P. freudenreichii vivant et métaboliquement active dans le côlon. Sa consommation réprime la production de la cytokine pro-inflammatoire IL-8 au niveau de la muqueuse intestinale porcine. Une approche mécanistique a révélé les rôles contrastés de différents composés de surface tels que les exopolysaccharides et les protéines de surface, dans l’immunomodulation. Certaines protéines sont impliquées dans la modulation du système immunitaire, alors que la capsule exopolysaccharidique annule cette modulation. Conclusions : Le rôle de chaque molécule est actuellement abordé dans un projet ANR (Sarter SURFace against INflammation of the Gut) par une approche multidisciplinaire mobilisant 7 partenaires. L’élucidation des mécanismes cellulaires et moléculaires induits, ainsi que les effets observés in vivo, ouvriront des perspectives pour l’utilisation de produits laitiers fermentés par P. freudenreichii répondant aux besoins spécifiques de populations ciblées.

The growth of Bacillus cereus group bacteria often limits the shelf-life of pasteurised liquid egg products and is also a putative toxin producer. This study was performed to better understand the route of contamination by B. cereus in egg products by studying the factors affecting eggshell contamination on-farm. 2. Eggs were collected in warm and cold seasons in 50 conventional laying farms in Western France. Egg surfaces were analysed for the presence of B. cereus group bacteria, environmental measurements were recorded and production practices were identified through a questionnaire filled out by the farmers. 3. A total of 44% of the farms were contaminated by mesophilic and 10% by psychrotrophic B. cereus group bacteria. No significant effect of the season was observed, whatever the thermal type. Several procedures were associated with reduced eggshell contamination by mesophilic bacteria, including the limitation of dust formation from manure and feeding and efficient disinfection of the silo, houses and the sanitary wall between houses. 4. The research highlights the need to promote prevention strategies, from farm to fork, for the control of eggshell and putative subsequent egg product contamination by B. cereus group bacteria.

The growth of Bacillus cereus group bacteria often limits the shelf-life of pasteurised liquid egg products and is also a putative toxin producer. This study was performed to better understand the route of contamination by B. cereus in egg products by studying the factors affecting eggshell contamination on-farm. 2. Eggs were collected in warm and cold seasons in 50 conventional laying farms in Western France. Egg surfaces were analysed for the presence of B. cereus group bacteria, environmental measurements were recorded and production practices were identified through a questionnaire filled out by the farmers. 3. A total of 44% of the farms were contaminated by mesophilic and 10% by psychrotrophic B. cereus group bacteria. No significant effect of the season was observed, whatever the thermal type. Several procedures were associated with reduced eggshell contamination by mesophilic bacteria, including the limitation of dust formation from manure and feeding and efficient disinfection of the silo, houses and the sanitary wall between houses. 4. The research highlights the need to promote prevention strategies, from farm to fork, for the control of eggshell and putative subsequent egg product contamination by B. cereus group bacteria.

Recent works have revealed highly strain-dependent immunomodulatory properties in Propionibacterium freudenreichii, a cheese starter bacterium (Foligné et al. 2010). The underlying mechanisms are unknown but preliminary attempts have shown that surface components are involved in these immunomodulatory effects. The most promising strains/components exert promising anti-inflammatory effects through induction of regulatory cytokines. The aim of this work is to identify key surface components of Propionibacterium freudenreichii responsible for the immunomodulatory properties. 12 strains of Propionibacterium freudenreichii were sorted according to their high or low regulatory properties, measured by induction of IL-10 cytokine in peripheral blood mononuclear cells stimulated by equal amounts of the different P. freudenreichii strains. These strains were sequenced by Illumina paired-end sequencing and de novo assembled using Velvet software. Genome sequences were automatically and manually annotated on the INRA-AGMIAL platform. Correlations are studied both (i) between quantitative traits (high or low induction of IL-10) and genotypic properties, namely presence/lack of in silico predicted proteins and (ii) between quantitative traits (high or low induction of IL-10) and in vitro data (surface exposed proteins). In both cases, we account for the phylogenetic inertia induced by the shared evolutionary history of the strains. For in vitro data, three methods (shedding using guanidium chloride, surface labeling using CyDye cyanine and shaving using trypsine combined with mass spectrometry) are used to identify P. freudenreichii surface proteins. Statistical analysis is used to highlight which of these proteins can potentially be involved in immunomodulation, comparing the proteins identified and IL-10 induction. Statistical analyses of both in vitro and in silico approaches based on genome sequencing reveal targets potentially involved in immunomodulation. Both strategies are complementary. Proteomic approach followed by statistical analysis allow the identification of surface exposed proteins associated with immunomodulation, whereas in silico approach point out the mechanisms explaining the presence/ absence of the candidates on bacterial surface. Genome sequences, either directly used (in silico approach) or used as database (in vitro approach) determine key genes involved in the studied phenotypes. Candidate genes identified in the present study will be further characterized and their role in immunomodulation confirmed by knock out or overexpression of selected genes.

Staphylococcus aureus is a major pathogen in ruminant mastitis. Staphylococcal mastitis in ovine hosts is remarkably variable in terms of acuteness and ranges from subclinical to gangrenous mastitis. S. aureus factors involved in such variations are still poorly documented. This study aimed at identifying the staphylococcal factors responsible for the acuteness of the infection. Two S. aureus strains, isolated from gangrenous (O11) or subclinical (O46) ewe mastitis, were selected for this work. Despite a close phylogenetic relationship, these strains were shown to reproducibly induce severe (O11) or mild (O46) symptoms in experimental ewe mastitis. Several omic approaches were combined to compare these two strains. Both strains were fully sequenced and their transcriptome and proteome were analyzed in culture conditions mimicking mastitis context. Results showed that dramatic differences between O11 and O46 rely on their ability to adapt to and to express their virulence in a mammary context rather than in their gene content. This study highlights differences in the expression of genes related to mobile genetic elements (especially prophages), the expression of genes involved in iron acquisition and metabolism, of transcriptional factors (SigmaB and SigmaS) and in exoprotein production. Serological Proteome Analysis (SERPA) showed that most of these exoproteins are indeed produced during mastitis. Analysis of 10 other S. aureus strains, focused on two of these exoproteins –SspB and a protein of unknown function- showed SspB is produced only in strains isolated from severe mastitis and the protein of unknown function in strains isolated from subclinical mastitis. The exact role of the genes identified here (exoproteins, mobile genetic elements, iron metabolism and acquisition) in the infection and in mastitis acuteness remains to be determined. Finally, we showed that O11 is transformable and can thus be subjected to mutagenesis. The genes identified here represent excellent candidates for targeted studies of the molecular basis of S. aureus pathogenesis in ruminant mastitis.

Staphylococcus aureus is an opportunistic pathogen in humans and warm-blooded animals. In milk production, it is a main cause of mastitis in dairy ruminants, which induces huge economical loss. In this work, S. aureus strains RF122 and Newbould305, responsible for severe or mild mastitis, were compared at the genome scale. Whole Genome PCR Scanning and Comparative Genome Hybridization showed that RF122 (ST151) was closely related to ovine strains whereas Newbould305 (ST115) co-clustered with bovine isolates (J.Bact. 2008. 190(19):6302). The goal of this work was to identify staphylococcal factors explaining the differences of clinical signs and mastitis acuteness observed during infections with these strains. We compared the two strains, in silico and in vitro, at the genome, proteome and phenotype levels. The genome of S. aureus Newbould305 was sequencedusing an Illumina Genome Analyzer. A total of 2752 coding sequences were detected and Newbould305 sequence was compared to that of RF122, which is publicly available. The overall Newbould305 genome shares high similarity with that of RF122. A majority of genes (n= 1819) were common to both strains. Proteome analysis of supernatant, cell wall and total cell fractions was carried out in conditions mimicking the mastitis context and results lead to an additional characterization at the phenotype level. Efficiency of adhesion and internalisation of both strains were tested and compared in vitro on bovine mammary epithelial cells (MAC-T). The values for adhesion and internalization showed significantly higher rates for Newbould305 with differences of up to 1 order of magnitude in adhesion and 1.5 orders of magnitude for internalization. However, once internalized, intracellular survival appeared identical for both strains. This work provides targets for further investigations, especially to determine whether the differences observed at these initial steps of infection are indeed critical for the development and severity of cow mastitis.

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Analise immunoproteomica de staphylococcus aureus causador da mastite ovina revela polipepideos diferencialmente reconhecidos por soros de animais infectados e portadores. 26 . Congresso Brasileiro de Microbiologia

Cette publication est également parue dans la revue "Journal of Animal Science", Vol.89, E-Suppl.1.; Staphylococcus aureus is a major cause of mastitis in ruminants. In ewe mastitis, symptoms range from subclinical to gangrenous mastitis. S. aureus factors or host-factors contributing to the different outcomes are not completely elucidated. In this study, experimental mastitis was induced on primiparous ewes using two S. aureus strains, isolated from gangrenous (strain O11) or subclinical (strain O46) mastitis. Strains induced drastically distinct clinical symptoms when tested in ewe and mice experimental mastitis. Notably, they reproduced mild (O46) or severe (O11) mastitis in ewes. Ewe sera were used to identify staphylococcal immunoreactive proteins commonly or differentially produced during infections of variable severity and to define core and accessory seroproteomes. Such SERological Proteome Analysis (SERPA) allowed the identification of 89 immunoreactive proteins, of which only 52 (58.4%) were previously identified as immunogenic proteins in other staphylococcal infections. Among the 89 proteins identified, 74 appear to constitute the core seroproteome. Among the 15 remaining proteins defining the accessory seroproteome, 12 were specific for strain O11, 3 were specific for O46. Distribution of one protein specific for each mastitis severity was investigated in ten other strains isolated from subclinical or clinical mastitis. We report here for the first time the identification of staphylococcal immunogenic proteins common or specific to S. aureus strains responsible for mild or severe mastitis. These findings open avenues in S. aureus mastitis studies as some of these proteins, expressed in vivo, are likely to account for the success of S. aureus as a pathogen of the ruminant mammary gland.

Staphylococcus aureus est un des pathogènes majeurs impliqués dans les mammites chez les ruminants. Les symptômes associés varient selon la sévérité des mammites (de la mammite subclinique à la mammite gangréneuse). L’origine de cette variabilité (virulence du pathogène/susceptibilité de l’hôte) n’est pas clairement établie à ce jour. Afin d’identifier les différences entre des souches de S. aureus impliquées dans des mammites de degrés de sévérité différents, nous avons comparé deux souches de S. aureus isolées de mammites gangréneuse (O11) et subclinique (O46) et reproduisant des tableaux cliniques distincts lors d’infections expérimentales en modèle brebis. Les deux souches ont été séquencées (Illumina) et leur transcriptome (puce pangénomique) et protéome (électrophorèse 2D) ont été analysés et comparés dans des conditions mimant le contexte mammite. O11 et O46 sont génotypiquement et génétiquement proches à un prophage (présent dans la souche O46) et quelques gènes présentant des insertions/délétions près. A l’inverse, les analyses protéomiques et transcriptomiques ont montré des différences majeures entre les deux souches. O11 produit plus de toxines et de protéases que la souche O46 mais moins de composés de surface (adhésines, capsule), quelle que soit la phase de croissance. Il est à noter que le système agr est fonctionnel et que son expression est identique dans les deux souches. Ces résultats ont été confirmés sur d’autres souches de S. aureus isolées de mammites ovines présentant des degrés de sévérité différents. De façon remarquable, nous avons observé plusieurs différences inter-souches dans des gènes impliqués dans le métabolisme du fer. Ainsi, O11 surexprime des gènes impliqués dans l’acquisition du fer (isdH, sbnC, opéron sir) alors que O46 présente en plus d’une sous expression de ces gènes, des délétions sur les gènes isdH et hrtB. L’expression de ces gènes chez les souches de S. aureus isolées de mammites pourrait être un élément clé dans le pouvoir infectieux au sein de la glande mammaire. Ces résultats montrent que, même si O11 et O46 ont en théorie un potentiel de virulence équivalent (comparaison génomique / contenu en gènes similaire), la capacité d’expression des facteurs de virulence ainsi que la capacité d’adaptation au contexte infectieux, (comparaison transcriptomique et protéomique) varient entre les deux souches et pourraient être à l’origine des degrés de sévérités distincts observés en contexte mammite

Staphylococcus aureus is a major cause of mastitis in ruminants. In ewe mastitis, symptoms range from subclinical to gangrenous mastitis. S. aureus factors or host-factors contributing to the different outcomes are not completely elucidated. In this study, experimental mastitis was induced on primiparous ewes using two S. aureus strains, isolated from gangrenous (strain O11) or subclinical (strain O46) mastitis. Strains induced drastically distinct clinical symptoms when tested in ewe and mice experimental mastitis. Notably, they reproduced mild (O46) or severe (O11) mastitis in ewes. Ewe sera were used to identify staphylococcal immunoreactive proteins commonly or differentially produced during infections of variable severity and to define core and accessory seroproteomes. Such SERological Proteome Analysis (SERPA) allowed the identification of 89 immunoreactive proteins, of which only 52 (58.4%) were previously identified as immunogenic proteins in other staphylococcal infections. Among the 89 proteins identified, 74 appear to constitute the core seroproteome. Among the 15 remaining proteins defining the accessory seroproteome, 12 were specific for strain O11, 3 were specific for O46. Distribution of one protein specific for each mastitis severity was investigated in ten other strains isolated from subclinical or clinical mastitis. We report here for the first time the identification of staphylococcal immunogenic proteins common or specific to S. aureus strains responsible for mild or severe mastitis. These findings open avenues in S. aureus mastitis studies as some of these proteins, expressed in vivo, are likely to account for the success of S. aureus as a pathogen of the ruminant mammary gland.

Staphylococcus aureus is a major pathogen involved in intra mammary infections (or mastitis) in ruminants. Accuteness of the disease vary from subclinical mastitis to gangrenous mastitis. The origin of such a variability depends on both S. aureus virulence and host susceptibility and is not clearly established yet.. Objective: Identify differences found in S. aureus strains inducing mastites with diverse acuteness in order to identify staphylococcal virulence markers. Method: Genome sequences of two S. aureus strains isolated from gangrenous mastitis (O11) and subclinical mastitis (O46) were obtained (Illumina). Both strains were grown in vitro in conditions mimicking a mastitis context, and their gene expression profile and proteome were analyzed and compared using comparative genomic hybridization and 2D gel electrophoresis. Results: O11 and O46 are genotypically and genetically closely related. Proteomic analysis of the two strains nevertheless revealed clear differences, notably in the exoprotein patterns (higher production of toxins such as lukM/F’-PV, alpha hemolysine, and glycolytic enzymes like enolase or adhesion proteins by O11 strain compared to O46). The genome sequence of the 2 strains allowed the identification of an additional phage in O46 strain, SNPs (1189 synonymous in 487 ORFs and 1104 non synonymous in 680 ORFs in the core genome) and indels (~80) in both strains. SNPs are scattered on the chromosome and involve all the functional categories including virulence factors, some of which harbor several SNPs. Transcriptomic analysis allowed to link genome and proteome data and explained some of the differences observed between the two strains. Conclusions Altogether, these results show that differences in terms of pathogenicity in S. aureus mastitis isolates can be correlated to gene content and, mainly, to the strain capability to express and produce virulence factors. Some genes were found specifically present or overexpressed in one strain or the other and could be involved to the differences observed in the mastitis acuteness. They would thus be interesting markers to characterize virulence potential of S. aureus strains isolated from ewe mastitis.

Staphylococcus aureus est un des pathogènes majeurs impliqués dans les infections intramammaires (mammites) chez les ruminants. Les symptômes associés varient selon la sévérité des mammites pouvant aller de la mammite subclinique à la mammite gangréneuse. L’origine de cette variabilité dans le degré de sévérité de la mammite (virulence du pathogène/susceptibilité de l’hôte) n’est pas encore clairement établie. Objectifs: Identifier les différences entre des souches de S. aureus impliquées dans des mammites à degrés de sévérité différents de façon à déterminer des marqueurs de virulence staphylococciques. Méthode: Deux souches de S. aureus isolées de mammites gangréneuse (O11) et subclinique (O46) ont été séquencées par la méthode Illumina. Après culture in vitro dans des conditions mimant le contexte mammite, leur transcriptome et protéome ont été analysés et comparés respectivement par hybridation sur puce et par électrophorèse bidimensionnelle. Résultats: O11 et O46 sont génotypiquement et génétiquement proches. L’étude protéomique de ces 2 souches a montré cependant des différences nettes, notamment au niveau des profils des exoprotéines (surproduction de toxines tels que lukM/F’-PV, alpha hémolysine, d’enzymes glycolytiques comme l’énolase ou de protéines d’adhésion par la souche O11 en comparaison de O46). Le séquençage des 2 souches a permis de mettre en évidence la présence d’un phage supplémentaire dans la souche O46, de SNPs (1189 synonymes concernant 487 ORFs et 1104 non synonymes concernant 680 ORFs au niveau du génome central) et d’indels (~50) dans les deux souches. Les SNPs sont répartis sur l’ensemble du génome et concernent toutes les catégories fonctionnelles y compris certains facteurs de virulence qui présentent un ou plusieurs SNPs. L’analyse transcriptomique globale a permis d’établir un lien entre génome et protéome et d'expliquer certaines différences observées entre les deux souches. Conclusions Dans l’ensemble, ces résultats montrent que les différences de potentiel pathogène observées sur des souches de S. aureus peuvent être corrélées à leur contenu en gènes et surtout à leur capacité à exprimer et à produire certains facteurs de virulence. Certains gènes spécifiquement présents ou surexprimés dans l’une des deux souches pourraient être à l’origine du degré de sévérité des mammites. Ils constitueraient alors des marqueurs du degré de virulence chez les souches de S. aureus associées aux mammites ovines