Systems biology applied to the resolution of variation of susceptibility to staphylococcal mastitis

Les mammites représentent encore à l'heure actuelle une des pathologies dominantes chez les ruminants laitiers. Ces inflammations de la glande mammaire sont caractérisées par une augmentation de la concentration des cellules, dites somatiques ou (CCS) dans le lait. En outre, il a été montré que le score de cellules somatiques, obtenu par transformation logarthmique de la valeur CCS (comptage de cellules somatiques) est bien corrélé à la présence d'une mammite clinique ou subclinique. Deux lignées divergentes de brebis ont été produites en utilisant les index génétique CCS de leurs parents. Ces animaux ont été caractérisés pour évaluer la différence de prédisposition aux infections mammaires. Ainsi, nous avons montré que ces animaux ont une différence significative de sensibilité aux mammites par les Staphylocoques. L'objectif de nos travaux est d'analyser les mécanismes qui sous-tendent les caractères de résistance/sensibilité chez les animaux issus de la sélection divergente. Nous avons tout d'abord recherché la présence d'une immunodépression des neutrophiles qui est differente entre les deux lignées. Dans ce but, la concentration des différentes cellules sanguines, la production de radicaux libres, et l'activité bactéricide dans la période pré- et post-partum ont été mésurées. Aucune différence n'a été mise en évidence entre les neutrophiles de la ligné sensible et résistante, bien qu'une altération significative des fonctions neutrophiliques a été constatée une semaine avant la mise bas et en période post-partum immédiat. Ainsi, nos résultats montrent que la différence de sensibilité aux infections mammaires ne dépend pas de l'altération des fonctions des neutrophiles sanguins autour de la mise bas (Article # 1). A l'étape suivante, dans l'objectif d'accroître notre connaissance des bases génétiques de la différence de réponse immunitaire aux infections staphylococciques, le transcriptome de cellules inflammatoires (principalement des neutrophiles) du lait a été analysé chez des animaux sensibles et résistants, infectés successivement par S. epidermidis et S. aureus. Malgré le fait que les deux bactéries sont génétiquement très proches, un grand nombre de gènes étaient différemment exprimés 12 heures après l'inoculation. En outre, cette analyse a confirmé une proportion plus grande de cellules T parmi les CCS, à la suite de l'infection par S. aureus. En outre, les 335 gènes différentiels entre la ligne sensible et la lignée résistante étaient principalement reliés à la réponse immunitaire et inflammatoire (incluant la voie de signalisation du récepteur aux pathogènes TLR-2 et la fonction de diapédèse, la prolifération cellulaire, et l'apoptose. Plus précisément, les marqueurs de l'inflammation (saa2, s100a2) étaient exprimés chez les animaux sensibles. Les données obtenues supportent l'idée que le recrutement des neutrophiles et l'adhésion cellulaire (alcam, il32, itga5, selp et st3gal4) est plus efficace chez les animaux résistants. De plus, un contrôle plus précoce de l'inflammation apparaît chez les animaux résistants, en relation avec un processus d'apoptose plus marqué (Article # 2). Enfin, pour mieux apprécier le rôle joué par différentes types cellulaires, une analyse du profil d'expression de cellules dendritiques générées in vitro a été réalisée après stimulation par S. aureus. Nous avons ainsi décrit le profil transcriptomique des DC stimulées par S .aureus ; les gènes codant pour les facteurs impliqués dans la réponse inflammatoire et les cytokines responsables de la polarisation T étaient très fortement sur-exprimés à la suite de la stimulation. Un groupe de 204 gènes était statistiquement différent entre les animaux sensibles et résistants ; le profil d'expression obtenu permet de grouper les animaux en fonction de leur groupe d'origine. Ces résultats montrent que les animaux sensibles ont une réponse inflammatoire plus marquée en relation avec une sur-expression des voies de signalisation du récepteur de l'IL-1 (IL1R). De plus, les fonctions relatives à la diapédèse sont dominantes dans la lignée sensible, alors que dans la lignée résistante, non seulement l'inflammation apparaît plus précocement, mais l'expression de gènes de la voie classique du complément (C1q) et l'indolamine-1 (ido1) indique la présence de phénomène de régulation (Article # 3). Nous avons clairement montré que les animaux sensibles et résistants ont des profils d'expression génique différents pour deux populations cellulaires majeures. L'analyse globale des gènes différentiels identifiés dans les neutrophiles et les cellules dendritiques suggèrent une inflammation plus marquée chez les animaux sensibles, alors que la régulation de l'inflammation apparaît plus rapidement chez les animaux résistants. L'analyse du transcriptome de ces deux types cellulaires chez des animaux avec des sensibilités aux infections staphylococciques différentes a permis d'identifier de nouveaux gènes candidats. Ainsi, de nouvelles voies biologiques impliquées dans la résistance associée à la sélection génétique sur les CCS ont été identifiées, et ces travaux ouvrent de nouvelles perspectives à explorer pour l'étude de la prédisposition aux infections staphylococciques.Mastitis is the most prevalent disease and major cause of economic losses in the dairy industry. Generally, mastitis is characterised as increase inflammatory cell count in the milk commonly called as Somatic cells concentration (SCC). Moreover, it has long been shown that somatic cell score (SCS) and clinical mastitis are positively correlated. Therefore, two lines of animals have been selected on the basis of extreme breeding values of their parents for SCS. This animal resource has been characterized for their potential difference of susceptibility for intramammary infections and it was noticed that animals have different degrees of susceptibility for Staphylococci infections. Global aim of the study was to investigate the mechanisms underlying resistance/susceptibility trait in the genetically-selected animals. Initially, we checked whether GNN functions are different in the two groups. We measured blood cell count, Reactive Oxygen Species (ROS) production after stimulation and bactericidal activity during the pre- and post-parturient period. We showed that no significant difference of ROS production and bactericidal activities of GNN exist between resistant and susceptible animals, but a decline in their activities do occur at parturition. Hence, our results indicate that difference of susceptibility to intramammary infections does not depend on alterations of blood GNN functions, at least at the beginning of lactation (Article # 1). In the next step, transcriptional profiling of inflammatory milk cells (MSC) of resistant and susceptible animals challenged by S. epidermidis and S. aureus successively was performed to enhance our understanding of the genetic basis of host responses to IMI. Despite the fact that both bacterial strains are quite closely related, a large number of genes were found differentially expressed at 12h-post inoculation. DE genes demonstrate that S. aureus induced an increased proportion of T cells in MSC. Moreover, the differential regulation of 335 genes between resistant and susceptible animals was mainly associated with the immune and inflammatory response, including the pathogen recognition TLR-2 pathway and cell recruitment, cell proliferation and apoptosis. More precisely, markers of inflammation (saa2, s100a2) were more expressed in the susceptible animals. Whereas, Toll like receptor (tlr2), antimicrobial peptides (cathl1b) diapedesis of neutrophils and cell adhesion (alcam, il32, itga5, selp and st3gal4) is more efficient in resistant animals. Moreover, a more efficient outcome of infection occurs in the resistant animals through efficient TLR-signalling and apoptotic process (Article # 2). To grab more insights into the immune system of these animals, transcriptional profiling of in vitro S. aureus-stimulated bone marrow-derived dendritic cells was performed. Gene signatures of stimulated DCs were obtained and genes involved in inflammatory process and T helper cell polarization were highly up-regulated upon stimulation. Moreover, a set of 204 genes were statistically different between susceptible and resistant animals, and grouped them according to their predisposition to Staphylococcal infection. We showed that in resistant animals, not only the process of inflammation is efficient but also the shutdown of inflammatory response through classical complement pathway (c1q) and early regulation of inflammation by IDO1 pathway (ido1) (Article # 3). We have clearly demonstrated that resistant and susceptible animals have distinct gene profiles of two major immune cell populations which correspond to their clinical picture and genetic phenotypes. Global analysis of DE expressed genes obtained from dendritic cells and inflammatory milk cells suggest that a more aggravated inflammation occurs in susceptible animals whereas in resistant animals not only an efficient onset of inflammation occurs but also a rapid regulation of inflammation does happen. Gene profiling of milk inflammatory cells and dendritic cells in resistant and susceptible animals has provided strong candidates for undermining the biological pathways involved in the somatic-cell based resistance trait, which open new avenues in the domain of mastitis to explore

Effectiveness of an antimicrobial treatment scheme in a confined glanders outbreak

BACKGROUND: Glanders is a contagious and fatal zoonotic disease of solipeds caused by the Gram-negative bacterium Burkholderia (B.) mallei. Although regulations call for culling of diseased animals, certain situations e.g. wild life conservation, highly valuable breeding stock, could benefit from effective treatment schemes and post-exposure prophylaxis. RESULTS: Twenty three culture positive glanderous horses were successfully treated during a confined outbreak by applying a treatment protocol of 12 weeks duration based on the parenteral administration of enrofloxacin and trimethoprim plus sulfadiazine, followed by the oral administration of doxycycline. Induction of immunosupression in six randomly chosen horses after completion of treatment did not lead to recrudescence of disease. CONCLUSION: This study demonstrates that long term treatment of glanderous horses with a combination of various antibiotics seems to eliminate the agent from the organism. However, more studies are needed to test the effectiveness of this treatment regime on B. mallei strains from different endemic regions. Due to its cost and duration, this treatment can only be an option in certain situations and should not replace the current “testing and culling” policy, in conjunction with adequate compensation to prevent spreading of disease

Differential Transcriptional Response To Staphylococcus aureus Infection In Two Divergent Lines Of Sheep Selected On Milk Somatic Cell Score

The milk somatic cell score (SCS) is an indirect indicator of mastitis, currently used as a selection criterion of dairy ruminants that leads to decreased intra-mammary infection prevalence. In the present study, gene expression profiles after Staphylococcus stimulations were determined in three cell types of mastitis resistant and susceptible ewes. The comparisons of the lists of the differentially-expressed genes allowed identification of commonly-regulated genes among cell types. These results lead to identify a subset of genes involved in pathogen-related receptor signaling. These genes may play an important role in recognition of pathogens and may improve resistance to intramammary infections

Transcriptomic analysis of milk somatic cells in mastitis resistant and susceptible sheep upon challenge with Staphylococcus epidermidis and Staphylococcus aureus

<p>Abstract</p> <p>Background</p> <p>The existence of a genetic basis for host responses to bacterial intramammary infections has been widely documented, but the underlying mechanisms and the genes are still largely unknown. Previously, two divergent lines of sheep selected for high/low milk somatic cell scores have been shown to be respectively susceptible and resistant to intramammary infections by <it>Staphylococcus spp</it>. Transcriptional profiling with an 15K ovine-specific microarray of the milk somatic cells of susceptible and resistant sheep infected successively by <it>S. epidermidis </it>and <it>S. aureus </it>was performed in order to enhance our understanding of the molecular and cellular events associated with mastitis resistance.</p> <p>Results</p> <p>The bacteriological titre was lower in the resistant than in the susceptible animals in the 48 hours following inoculation, although milk somatic cell concentration was similar. Gene expression was analysed in milk somatic cells, mainly represented by neutrophils, collected 12 hours post-challenge. A high number of differentially expressed genes between the two challenges indicated that more T cells are recruited upon inoculation by <it>S. aureus </it>than <it>S. epidermidis</it>. A total of 52 genes were significantly differentially expressed between the resistant and susceptible animals. Further Gene Ontology analysis indicated that differentially expressed genes were associated with immune and inflammatory responses, leukocyte adhesion, cell migration, and signal transduction. Close biological relationships could be established between most genes using gene network analysis. Furthermore, gene expression suggests that the cell turn-over, as a consequence of apoptosis/granulopoiesis, may be enhanced in the resistant line when compared to the susceptible line.</p> <p>Conclusions</p> <p>Gene profiling in resistant and susceptible lines has provided good candidates for mapping the biological pathways and genes underlying genetically determined resistance and susceptibility towards <it>Staphylococcus </it>infections, and opens new fields for further investigation.</p

Genetic susceptibility to S. aureus mastitis in sheep: differential expression of mammary epithelial cells in response to live bacteria or supernatant

Staphylococcus aureus is a prevalent pathogen for mastitis in dairy ruminants and is responsible for both clinical and subclinical mastitis. Mammary epithelial cells (MEC) represent not only a physical barrier against bacterial invasion but are also active players of the innate immune response permitting infection clearance. To decipher their functions in general and in animals showing different levels of genetic predisposition to Staphylococcus in particular, MEC from ewes undergoing a divergent selection on milk somatic cell count were stimulated by S. aureus. MEC response was also studied according to the stimulation condition with live bacteria or culture supernatant. The early MEC response was studied during a 5 h time course by microarray to identify differentially expressed genes with regard to the host genetic background and as a function of the conditions of stimulation. In both conditions of stimulation, metabolic processes were altered, the apoptosis-associated pathways were considerably modified, and inflammatory and immune responses were enhanced with the upregulation of il1a, il1b, and tnfa and several chemokines known to enhance neutrophil (cxcl8) or mononuclear leukocyte (ccl20) recruitment. Genes associated with oxidative stress were increased after live bacteria stimulation, whereas immune responserelated genes were higher after supernatant stimulation in the early phase. Only 20 genes were differentially expressed between Staphylococcus spp-mastitis resistant and susceptible animals without any clearly defined role on the control of infection. To conclude, this suggests that MEC may not represent the cell type at the origin of the difference of mastitis susceptibility, at least as demonstrated in our genetic model. Supernatant or heat-killed S. aureus produce biological effects that are essentially different from those induced by live bacteria

Gene expression profiling of dendritic cells reveals important mechanisms associated with predisposition to Staphylococcus infections.

BACKGROUND: Staphylococcus aureus is a major pathogen of humans and animals and emerging antibiotic-resistant strains have further increased the concern of this health issue. Host genetics influence susceptibility to S. aureus infections, and the genes determining the outcome of infections should be identified to find alternative therapies to treatment with antibiotics. Here, we used outbred animals from a divergent selection based on susceptibility towards Staphylococcus infection to explore host immunogenetics. METHODOLOGY/PRINCIPAL FINDINGS: We investigated how dendritic cells respond to heat-inactivated S. aureus and whether dendritic cells from animals showing different degrees of susceptibility had distinct gene expression profiles. We measured gene expression levels of in vitro S. aureus-stimulated bone marrow-derived dendritic cells at three different time points (0, 3 and 8 hrs) by using 15 k ovine Agilent microarrays. Furthermore, differential expression of a selected number of genes was confirmed by RT-qPCR. Gene signatures of stimulated DCs were obtained and showed that genes involved in the inflammatory process and T helper cell polarization were highly up-regulated upon stimulation. Moreover, a set of 204 genes were statistically differentially expressed between susceptible and resistant animals, and grouped them according to their predisposition to staphylococcal infection. Interestingly, over-expression of the C1q and Ido1 genes was observed in the resistant line and suggested a role of classical pathway of complement and early regulation of inflammation pathways, respectively. On the contrary, over expression of genes involved in the IL1R pathway was observed in susceptible animals. Furthermore, the leucocyte extravasation pathway was also found to be dominant in the susceptible line. CONCLUSION/SIGNIFICANCE: We successfully obtained Staphylococcus aureus associated gene expression of ovine BM-DC in an 8-hour kinetics experiment. The distinct transcriptional profiles of dendritic cells obtained from resistant and susceptible animals may explain susceptibility towards S. aureus infections in a broader context

Effectiveness of an antimicrobial treatment scheme in a confined glanders outbreak

Abstract Background Glanders is a contagious and fatal zoonotic disease of solipeds caused by the Gram-negative bacterium Burkholderia (B.) mallei. Although regulations call for culling of diseased animals, certain situations e.g. wild life conservation, highly valuable breeding stock, could benefit from effective treatment schemes and post-exposure prophylaxis. Results Twenty three culture positive glanderous horses were successfully treated during a confined outbreak by applying a treatment protocol of 12 weeks duration based on the parenteral administration of enrofloxacin and trimethoprim plus sulfadiazine, followed by the oral administration of doxycycline. Induction of immunosupression in six randomly chosen horses after completion of treatment did not lead to recrudescence of disease. Conclusion This study demonstrates that long term treatment of glanderous horses with a combination of various antibiotics seems to eliminate the agent from the organism. However, more studies are needed to test the effectiveness of this treatment regime on B. mallei strains from different endemic regions. Due to its cost and duration, this treatment can only be an option in certain situations and should not replace the current “testing and culling” policy, in conjunction with adequate compensation to prevent spreading of disease.</p