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Antibiotic resistance has been reported since the 1940s in both human and veterinary medicine. Many years of monitoring milk samples in South Africa led to identification of a novel maltose-negative Staphylococcus aureus (S. aureus) strain, which appears to be an emerging pathogen. In this study, the susceptibility of this strain to antibiotics was evaluated over time, during diverse seasons in various provinces and according to somatic cell count (SCC) categories. A data set of 271 maltose-negative S. aureus isolates, from milk samples of 117 dairy herds, was examined using the disk diffusion method, between 2010 and 2017. This study also compared the susceptibility testing of 57 maltose-negative and 57 maltose-positive S. aureus isolated from 38 farms, from three provinces using minimum inhibitory concentration (MIC). The MIC results for the maltose-negative S. aureus isolates showed highest resistance to ampicillin (100%) and penicillin (47.4) and lowest resistance (1.8%) to azithromycin, ciprofloxacin and erythromycin. The maltose-negative S. aureus isolates showed overall significantly increased antibiotic resistance compared to the maltose-positive strains, as well as multidrug resistance. Producers and veterinarians should consider probability of cure of such organisms (seemingly non-chronic) when adapting management and treatment, preventing unnecessary culling.

National audience; Six vaches de race Prim’Holstein sont traitées au tarissement avec un produit intramammaire hors-lactation à base de cloxacilline (1 seringue par quartier). Le lendemain, les 6 vaches présentent une hyperthermie et une inflammation sévère des 4 quartiers.

Exfoliative toxins (ETs) are secreted virulence factors produced by staphylococci. These serine proteases specifically cleave desmoglein 1 (Dsg1) in mammals and are key elements in staphylococcal skin infections. We recently identified a new et gene in S. aureus O46, a strain isolated from ovine mastitis. In the present study, we characterized the new et gene at a genetic level and the enzymatic activity of the deduced protein. The S. aureus O46 genome was re-assembled, annotated and compared with other publicly available S. aureus genomes. The deduced amino acid sequence of the new et gene shared 40%, 53% and 59% sequence identity to those of ETA, ETB and ETD, respectively. The new et gene shared the same genetic vicinity and was similar in other S. aureus strains bearing this gene. The recombinant enzyme of the new et gene caused skin exfoliation in vivo in neonatal mice. The new et-gene was thus named ete, encoding a new type (type E) of exfoliative toxin. We showed that ETE degraded the extracellular segments of Dsg1 in murine, ovine and caprine epidermis, as well as in ovine teat canal epithelia, but not that in bovine epidermis. We further showed that it directly hydrolyzed human and swine Dsg1 as well as murine Dsg1α and Dsg1β, but not canine Dsg1 or murine Dsg1γ. Molecular modeling revealed a correlation between the preferred orientation of ETE docking on its Dsg1 cleavage site and species-specific cleavage activity, suggesting that the docking step preceding cleavage accounts for the ETE species-specificity. This new virulence factor may contribute to the bacterial colonization on the stratified epithelia in certain ruminants with mastitis.

Vésicules extracellulaires de [i]Staphylococcus aureus[/i]. Composition et rôle en contexte mammite. 1ère Rencontre Op+Lait-INRA

Dans cette thèse, nous nous sommes intéressée à une mutation dans le gène SOCS2(Suppressor Of Cytokine Signaling 2) dont l’effet est pléiotrope, c’est-à-dire qu’il concerne plusieurs caractères d’intérêt chez la brebis laitière Lacaune. Cette mutation estdéfavorable pour la résistance aux mammites (inflammation de la mamelle), et favorable pour les caractères de croissance et de productionlaitière. Située dans le domaine de liaison très conservé de la protéine SOCS-2, cette mutation ponctuelle (SNP:Single Nucleotide Polymorphism) entraîne la perte de fonctionnalité de la protéine qui est impliquée dans une voie majeure de signalisation chez les mammifères, la voie JAK (Janus Kinase) / STAT (Signal Transducer and Activator of Transcription), régulant un large spectre de cytokines et de facteurs de croissance.Le premier objectif de cette thèse était de proposer la prise en compte de cette mutation dans le contexte de la sélection génomique dans la race Lacaune. Les méthodes d’évaluation pondérées, et l’ajout du SNP SOCS2parmi les marqueurs de la puce, ont apporté des gains de précisions sur les prédictions (respectivement +3,99% et +0,26% en moyenne), laissant envisager la possibilité d’unesélection plus efficace. La méthode Gene Contentétait également intéressante car elle a permis de dissocier la valeur génétique due au gène SOCS2de celle provenant des autres gènes (polygène). Ainsi nous avons montré que la sélection actuelle en race Lacaune permet de diminuer la fréquence de l’allèle défavorable SOCS2tout en améliorant la résistance aux mammites expliquée par le reste du génome.Le deuxième objectif était d’étudier les effets de SOCS-2 sur des caractères d’intérêt non évalués jusqu’à présent: statut infectieux des mamelles et reproduction chez la femelle, et croissance des mâles. Des études d’association avec une puce de 960 SNP, dont le SNP SOCS2, ont confirmé l’effet de SOCS2sur la production laitière et sur l’inflammation mammaire, et ont montré un effet direct sur l’infection à l’aide de nouveaux phénotypes fins (bactériologie, examens cliniques). Des modèles linéaires ont montréque la mutation SOCS2était associée à une augmentation de poids chez les jeunes mâles (+1,5%), comme cela avait été observé chez la brebis, et àune diminution du taux de réussite à l’insémination artificielle chez les brebis (+1,3 fois de risque d’échec), sans effet sur la prolificité. Ces résultats marquants et originaux sur la reproduction ouvrent la voie à des analyses complémentaires pour déterminer notamment à quelstade biologique (ovulation, fécondation, implantation embryonnaire, etc.) la protéine SOCS-2 pourrait intervenir.Le dernier objectif était de déterminer le rôle de la protéine SOCS-2 dans les mécanismes biologiques sous-jacents de l’immunité grâce à l’étude de la réponse de 14 brebis porteuses et non porteuses de la mutation à l’état homozygote dans le cadre d’une infection intra-mammaire par un Staphylococcus aureus. L’ensemble des brebis a présenté deux pics de recrutement de cellules immunitaires versla mamelle à 20 et 88 heures post-inoculation (T20 et T88). Une analyse différentielle des gènes exprimés a confirmé une modification du transcriptome à partir de T16 en lien avec l’activation du système immunitaire,des voies de réparation de l’ADN et d’apoptose cellulaire. Les brebis portant la mutation SOCS2se sont différenciées des brebis sauvages, par une réaction clinique accrue à T56 et une différence de recrutement de lymphocytes T au moment des pics inflammatoires. A T56, un total de 177 gènes était différentiellement exprimé entre les deux génotypes. Une analyse fonctionnelle de ces gènes, et l’intégration entre l’expression génique et les phénotypes, suggèrent que la mutation conduit à une sur activation des voies de transmission de signal et derégulation (interférons, STAT3) avec un impact délétère sur l’état clinique des brebis., In this PhD thesis, we investigated a mutation in the SOCS2gene (Suppressor Of Cytokine Signaling 2) with a pleiotropic effect, i.e.it affects several traits of interest in the Lacaune dairy sheep. This mutation is unfavorable for mastitis resistance (udderinflammation), and favorable for growth and milk production traits.This point mutation (SNP -Single Nucleotide Polymorphism), located in the highly conserved binding domain of the SOCS-2 protein,causes the loss of functionality of the protein that is involved in a major signaling pathway in mammals:the JAK(Janus Kinase) / STAT (Signal Transducer and Activator of Transcription) pathway, regulating a large spectrum of cytokines and growth factors.The first objective of this PhD thesis was to provide a basis for considering this mutation in the context of genomic selection in the Lacaune breed. Weighted evaluation methods, and the addition of the SOCS2SNP among the chip markers, brought gains in accuracy on predictions (+3.99% and +0.26% respectively on average), suggesting the possibility of more efficient selection. The Gene Contentmethod was also interesting because it alloweddissociatingthe genetic value due to the SOCS2gene from that of the other genes (polygene). Thus, we have shown that the current selection in Lacaune breed allows reducing the frequency of the unfavorable SOCS2allele while improving the resistance to mastitis explained by the remaining part of the genome.The second objective was to study the effects of SOCS2on traits of interest not yet investigated: udder infection status and reproduction in females, and growth in males. Association studies with a 960 SNP chip, including SOCS2SNP, confirmed the effect of SOCS2on milk production and mammary inflammation, and showed a direct effect on infection using new fine phenotypes (bacteriology, clinical examinations). Linear models showed that the SOCS2mutation was associated withan increase in weight in young males (+1.5%), as observed in ewes, and a decrease in the artificial insemination success rate in ewes (+1.3 times the risk of failure), with no effect on prolificacy. These striking and original results on reproduction pave the way for further analyses to determine in particular at what biological stage (ovulation, fecundation, embryonic implantation, etc.) the SOCS-2 protein could be involved.The last objective was to determine the role of the SOCS-2 protein in the underlying biological mechanisms of immunity by investigating the response of 14 homozygous carrier and non-carrier ewes to intramammary infection by Staphylococcus aureus. All ewes developed two peaks of immune cell recruitment to the udder at 20 and 88 hours post-inoculation (T20 and T88). Differential analysis of the expressed genes confirmed a modification of the transcriptome from T16 related to the activation of the immune system, the DNA repair and the cellular apoptosis pathways. Ewes carrying the SOCS2mutation were distinguished from wild ewes by an increased clinical response at T56 and a difference in T lymphocyte recruitment at the time of the inflammatory peaks. At T56, a total of 177 genes were differentially expressed between the two genotypes. A functional analysis of these genes, and the integration between gene expression and phenotypes, suggest that the mutation leads to an over activation of signal transmission and regulatory pathways (interferons, STAT3) with a deleterious impact on the clinical status of ewe.

Mastitis, an inflammation of the mammary gland, is the most prevalent production disease in dairy herds worldwide. It affects animal health and milk quality and causing huge economic losses. Staphylococcus aureus is one of the most important etiological agents of mastitis, and treatment against S. aureus-induced mastitis is still ineffective. Although S. aureus is extensively studied, the mechanisms involved in its pathogenicity are not yet fully understood. Since secretory factors are important components in S. aureus pathogenesis, the role of extracellular vesicles (EVs) in infections is an emerging field of research in S. aureus. EVs are indeed nano-sized biological particles ranging from 30 to 150 nm and that are actively secreted into the surrounding environment by any living cell. They play a crucial role in intercellular communication and vectorization of bioactive molecules, and can modulate the metabolism and physiology of target cells, participating in pathogenesis, modulation of the immune response and exchange of material. Various virulence factors have been identified in S. aureus EVs. Additionally, EVs secreted by S. aureus N305 strain, a bovine mastitis isolate, induced the expression of IL-8 in vitro, and promoted tissue inflammation, deterioration, and local cytokine production, in vivo, suggesting a role of EVs in mastitis pathogenesis. The objective of this work is to investigate the involvement of S. aureus EVs in the host-pathogen interactions at the cellular and molecular levels, with special attention to the modulation of cellular and immune response. For that, an RNA-seq approach was chosen to identify eukaryotic genes and signaling pathways targeted and triggered by S. aureus N305 EVs in vitro, which is ongoing. Afterwards, the expression of relevant genes activated in vitro will be measured in mammary gland tissues exposed to EVs to confirm the mechanisms of action of EVs in vivo. This work will improve our knowledge on the interactions of EVs with host cells, bringing information about S. aureus mastitis pathogenesis. Additionally, it will also provide molecular and functional data for the use of EVs as biotechnological agents, e.g. for the development of vaccines or drugs for mastitis prevention.

Milk microbiota: physiological and health implication.. Meeting du LIA Bact-Inflam

Inflammation, DNA damage and Staphylococcus aureus infection. Meeting du LIA Bact-Inflam

International audience; Mastitis remains an important disease in dairy farming nowadays. Genetic selection of animals for increased resistance to mastitis is a relevant strategy to reduce the burden that mastitis entails on dairy cows. A divergent selection scheme based on somatic cell counts and clinical mastitis records was set-up on Prim’Holstein cows at the INRA experimental unit of Le Pin-au-Haras. In order to better define the mechanism underlying genetic resistance to mastitis, inflammatory challenges as well as immunization with a model antigen were performed on cows from these two different lines. LPS was infused in one healthy udder quarter of each cow approx. 1 month post-partum. Milk was collected 4, 8, 12 and 24h post-infusion. Somatic cells recruited in milk were counted and the cytokines/chemokines CXCL8, IL-6 and IL-1β were measured by ELISA. For adaptive response studies, cows were immunized 15 days before dry-off with ovalbumin and the response was evaluated 15 days after dry-off. Response to immunization was evaluated by measuring IFNγ and IL-17A and upon antigen stimulation in whole blood assays. Although LPS triggered mastitis in all infused quarters, mastitis resistant cows (n= 16) showed decreased inflammatory response compared to more susceptible cows (n= 25) at t=8h post-infusion. No differences were observed between the two groups in terms of response to immunization. Altogether, these results suggest that, in response to intra-mammary LPS challenge, cows selected for resistance to mastitis are better able to control the inflammatory response.

National audience

Microbial approaches againts bovine mastitis in dairy cows. STLOpendays

Milk somatic cell count (SCC) is commonly higher in goats than in cattle and sheep. Furthermore, the ability of milk SCC to predict mastitis is considered lower in goats than in cattle and sheep, and the relevance of somatic cell score (SCS)-based selection in this species has been questioned. To address this issue, we created 2 divergent lines of Alpine goats using artificially inseminated bucks with extreme estimated breeding values for SCS. A total of 287 goats, 158 in high- and 129 in low-SCS lines, were scrutinized for mastitis infections. We subjected 2,688 milk samples to conventional bacteriological analyses on agarose and bacterial counts were estimated for positive samples. The SCS, milk yield, fat content, and protein content were recorded every 3 wk. Clinical mastitis was systematically noted. A subset of 40 goats (20 from each line) was subsequently challenged with Haemonchus contortus and monitored for anemia (blood packed cell volume) and fecal egg counts to see if SCS-based selection had an indirect effect on resistance to gastrointestinal nematodes. Milk production traits, including milk quantity, fat content, and protein content, were similar in both goat lines. In contrast, the raw milk SCC almost doubled between the lines, with 1,542,000 versus 855,000 cells/mL in the high- and low-SCS lines, respectively. The difference in breeding value for SCS between lines was 1.65 genetic standard deviation equivalents. The Staphylococcus spp. most frequently isolated from milk were S. xylosus, S. caprae, S. epidermidis, and S. aureus. The frequency of positive bacteriology samples was significantly higher in the high-SCS line (49%) than in the low-SCS line (33%). The highest odds ratio was 3.49 (95% confidence interval: 11.95-6.25) for S. aureus. The distribution of bacterial species in positive samples between lines was comparable. The average quantity of bacteria in positive samples was also significantly higher in high-SCS goats (69 ± 80 growing colonies) than in low-SCS goats (38 ± 62 growing colonies). Clinical cases were rare and equally distributed between high- (n = 4; 2.5%) and low-SCS (n = 3; 2.3%) lines. Furthermore, the larger the amounts of bacteria in milk the higher the SCS level. Conversely, goats with repeatedly culture-negative udders exhibited the lowest SCC levels, with an average of below 300,000 cells/mL. We therefore confirmed that SCS is a relevant predictor of intramammary infection and hygienic quality of milk in goats and can be used for prophylactic purposes. After challenge with H. contortus, goats were anemic with high fecal egg counts but we found no difference between the genetic lines. This result provides initial evidence that resistance to mastitis or to gastrointestinal nematodes infections is under independent genetic regulation. Altogether, this monitoring of the goat lines indicated that SCS-based selection helps to improve udder health by decreasing milk cell counts and reducing the incidence of infection and related bacterial shedding in milk. Selection for low SCC should not affect a goat's ability to cope with gastrointestinal nematodes.

International audience; Interleukin 17A-producing T helper cells (Th17) are effector memory CD4+ T cells that are crucial to adaptive immunity to extracellular bacteria. The activities of these cells in the bovine species are not yet defined for want of straightforward cultivation and isolation procedures. We have developed a method to cultivate, expand, sort and maintain in culture bovine Th17 cells from circulating CD4+ T cells of adult cows. Using polyclonal stimulation (antibodies to CD3 and CD28), we expanded positive CD4+ IL-17A+ T (Th17) cells in a cell culture medium without serum supplemented with TGF-β1 and IL-6. We used IL-2 to expand the cells, which were characterized by intracellular labeling for IL-17A and IFN-γ. Then, we isolated populations of CD4+ T cells producing IL-17A, IFN-γ or both by labeling surface IL-17A with either a complex of biotinylated anti-CD45 antibodies-streptavidin-biotinylated anti-IL-17A antibodies or by direct surface labeling with antibodies to IL-17A, followed by flow cytometry cell sorting. The percentages of surface-labeled IL-17A-secreting cells were quite similar to the percentages of intracellular-labeled IL-17A-producing cells of the same cultures, and the two labeling procedures of live cells yielded similar results. The sorted IL-17A+ cells were restimulated and expanded. After expansion, 80% of the isolated Th17 cells were positive for IL-17A intracellular labeling. The sorted IL-17A+ cells can be frozen, stored and expanded again. The sorted Th17 cells secreted much more IL-17A and IL-17F than did CD4+ IL-17- cells. Notably, most of Th17 cells secreted IFN-γ, although in lower amounts than did CD4+ IL-17- cells. Sorted cells were characterized by transcriptomic profiling. Genes coding for Th17 signature cytokines (IL-17A, IL-17F, IL-26) and transcription factors (RORγt, RORa) were overexpressed in Th17 cells. The techniques developed will make it possible to investigate the phenotypic and functional profiles of bovine Th17 cells along with their stability or plasticity.

Mastitis remains an important disease in dairy farming nowadays. Genetic selection of animals for increased resistance to mastitis is a relevant strategy to reduce the burden that mastitis entails on dairy cows. A divergent selection scheme based on somatic cell counts and clinical mastitis records was set-up on Prim’Holstein cows at the INRA experimental unit of Le Pin-au-Haras. In order to better define the mechanism underlying genetic resistance to mastitis, inflammatory challenges as well as immunization with a model antigen were performed on cows from these two different lines. LPS was infused in one healthy udder quarter of each cow approx. 1 month post-partum. Milk was collected 4, 8, 12 and 24h post-infusion. Somatic cells recruited in milk were counted and the cytokines/chemokines CXCL8, IL-6 and IL-1β were measured by ELISA. For adaptive response studies, cows were immunized 15 days before dry-off with ovalbumin and the response was evaluated 15 days after dry-off. Response to immunization was evaluated by measuring IFNγ and IL-17A and upon antigen stimulation in whole blood assays. Although LPS triggered mastitis in all infused quarters, mastitis resistant cows (n= 16) showed decreased inflammatory response compared to more susceptible cows (n= 25) at t=8h post-infusion. No differences were observed between the two groups in terms of response to immunization. Altogether, these results suggest that, in response to intra-mammary LPS challenge, cows selected for resistance to mastitis are better able to control the inflammatory response.

Exfoliative toxins (ETs) are secreted virulence factors produced by staphylococci. These serine proteasesspecifically cleave desmoglein 1 (Dsg1) in mammals and are key elements in staphylococcal skininfections. We recently identified a new et gene in S. aureus O46, a strain isolated from ovine mastitis.In the present study, we characterized the new et gene at a genetic level and the enzymatic activity ofthe deduced protein. The S. aureus O46 genome was re-assembled, annotated and compared with otherpublicly available S. aureus genomes. The deduced amino acid sequence of the new et gene shared 40%,53% and 59% sequence identity to those of ETA, ETB and ETD, respectively. The new et gene sharedthe same genetic vicinity and was similar in other S. aureus strains bearing this gene. The recombinantenzyme of the new et gene caused skin exfoliation in vivo in neonatal mice. The new et-gene wasthus named ete, encoding a new type (type E) of exfoliative toxin. We showed that ETE degraded theextracellular segments of Dsg1 in murine, ovine and caprine epidermis, as well as in ovine teat canalepithelia, but not that in bovine epidermis. We further showed that it directly hydrolyzed human andswine Dsg1 as well as murine Dsg1α and Dsg1β, but not canine Dsg1 or murine Dsg1γ. Molecularmodeling revealed a correlation between the preferred orientation of ETE docking on its Dsg1 cleavagesite and species-specific cleavage activity, suggesting that the docking step preceding cleavageaccounts for the ETE species-specificity. This new virulence factor may contribute to the bacterialcolonization on the stratified epithelia in certain ruminants with mastitis.

The role of the recently described interleukin-32 (IL-32) in Staphylococcus aureus -induced mastitis, an inflammation of the mammary gland, is unclear. We determined expression of IL-32, IL-6, and IL-8 in S. aureus - and Escherichia coli -infected bovine mammary gland epithelial cells. Using live bacteria, we found that in S. aureus -infected cells, induction of IL-6 and IL-8 expression was less pronounced than in E. coli -infected cells. Notably, IL-32 expression was decreased in S. aureus -infected cells, while it was increased in E. coli -infected cells. We identified the staphylococcal phenol-soluble modulin (PSM) peptides as key contributors to these effects, as IL-32, IL-6, and IL-8 expression by epithelial cells exposed to psm mutant strains was significantly increased compared to that in cells exposed to the isogenic S. aureus wild-type strain, indicating that PSMs inhibit the production of these interleukins. The use of genetically complemented strains confirmed this observation. Inasmuch as the decreased expression of IL-32, which is involved in dendritic cell maturation, impairs immune responses, our results support a PSM-dependent mechanism that allows for the development of chronic S. aureus -related mastitis.

Staphylococcus aureus (S. aureus) is a major pathogen involved in ruminant mastitis and present worldwide. Clinical signs of S. aureus mastitis vary considerably and are largely dependent on strain-specific factors. A comparison of two S. aureus strains that reproducibly induced either severe (O11) or mild (O46) mastitis in ewes revealed that the transcriptional regulator sigS was mutated in O46 (Le Maréchal et al., 2011. PLoS One. 6 (11) e27354. doi:10.1371/journal.pone.0027354). In the present paper, we analysed the sigS sequence in 18 other S. aureus strains isolated from goat or ewe mastitis and found a 4-bp deletion similar to that of the O46 sigS gene in three strains associated with subclinical ewe mastitis. This sigS gene was disrupted in strain O11 (O11ΔsigS), so our aim was to investigate its involvement in the severity of infections in the context of mastitis. The wild type (wt) and mutant strains were then characterized in vitro to determine the involvement of sigS in the response S. aureus under various stress conditions, and assess its influence on the cytotoxicity of the pathogen, its invasive capacity and biofilm formation. The strains were compared in vivo in an experimental mouse mastitis model in which clinical signs and cytokine production were evaluated at 24 h post-infection. While no significant differences in the effect on bacterial growth between O11 and O11ΔsigS were observed either in vitro or in vivo, a significantly weaker in vivo production of interleukin (IL)-1α, IL-1β, and Tumor Necrosis Factor (TNF)-α was measured in the mammary glands infected with the mutant strain, suggesting that infection with O11ΔsigS induced an attenuated local innate immune response. These results suggest an impact of sigS disruption on S. aureus pathogenesis in a ruminant mastitis context. This disruption is probably involved in, and may partly explain, the milder symptoms previously observed in S. aureus O46-induced mastitis in ewes.

Pendant la période de transition chez les vaches laitières, la demande énergétique pour initier la production de lait est très élevée, tandis que l’apport d’énergie est faible, ce qui crée inévitablement une balance énergétique négative. Ce déséquilibre physiologique constitue l'un des principaux facteurs de risque dans le développement de maladies de transition, et ensemble, ils peuvent influencer les performances reproductrices et augmenter le taux de réforme. De nouvelles stratégies, aidant les vaches laitières à supporter une production intensive, doivent donc être mises en place. Bien que des progrès remarquables aient été réalisés pour garantir un apport énergétique élevé, peu de recherches ont évalué la possibilité de contrôler les dépenses énergétiques. La traite incomplète des vaches en début de lactation pourrait aider à limiter le déséquilibre énergétique chez les vaches laitières. L'objectif de ce projet était de mesurer l'impact, sur la santé et la production, d'une traite incomplète durant les cinq premiers jours en lait (JEL ; sans changer la fréquence de traite), sur les vaches de fermes laitières commerciales. Plus spécifiquement, les éléments suivants ont été évalués : la production de lait, le taux de réforme, l'incidence de maladies infectieuses, les performances reproductrices et la sensibilité au niveau du pis. Un essai contrôlé randomisé a été réalisé chez les vaches multipares provenant d’un échantillon de convenance de 13 fermes laitières. Dans chaque troupeau, toutes les vaches multipares (n = 878), ayant mis bas entre décembre 2013 et mars 2015, ont été aléatoirement réparties entre un groupe traitement et un groupe témoin, à l'aide d'un générateur de nombres aléatoires. Les vaches du groupe traitement ont été soumises à une traite incomplète durant les cinq premiers JEL, avec une collecte maximale de 10, 10, 10, 12 et 14 litres de lait par jour aux JEL un, deux, trois, quatre et cinq, respectivement. Les vaches du groupe témoin ont été traites de manière conventionnelle. Le taux de réforme des vaches et la production de lait ne différaient pas entre les deux groupes. Lorsqu'on s'intéresse aux différences de rendement en termes de lait corrigé en énergie, les vaches traites incomplètement produisaient de façon similaire aux vaches traites complètement. La traite incomplète n'affectait pas les cotes de nouvelles infections intramammaires du 11 au 18ème JEL, ni les cotes de maladies du système reproducteur à 35 JEL, ni l'incidence de mammite clinique durant les 90 premiers JEL. Les cotes d'élimination d’une infection intramammaire du 11 au 18ème JEL chez les vaches traites incomplètement étaient 2,9 fois supérieures à celles des vaches traites complètement (intervalle de confiance à 95% : 1,4-6,0). La traite incomplète n'affectait pas non plus l'activité lutéale ; elle avait, cependant, un impact positif sur le risque de conception chez les vaches en deuxième lactation qui étaient dans des troupeaux avec une période d’attente volontaire inférieure à 55 jours (180/775 vaches). Chez ces vaches, le risque de conception (intervalle de confiance à 95%) pour les vaches traites incomplètement était 576,3 (240,0-1383,7), 36,9 (18,9-72,1), 6,8 (3,3-13,8), 2,5 (1,0-5,9), et 0,13 (0,07-0,26) fois celui des vaches traites normalement à 1-21, 22-43, 44-65, 66-87 et >87 jours respectivement après la période d'attente volontaire. L'algomètre de pression a été validé pour mesurer des changements de sensibilité dus à une distension du pis. Cet instrument était modérément répétable pour quantifier le seuil nociceptif mécanique sur le pis et de nombreux facteurs externes influençaient également les valeurs obtenues. Par conséquent, son utilisation pour cet usage devrait être considéré avec prudence. Nous avons donc plutôt observé le comportement de repos pour évaluer une douleur éventuelle ressentie au niveau du pis des vaches traites de façon incomplète. Nous n'avons observé aucun effet sur le temps de repos. Cependant, l'impact de la traite incomplète sur la fréquence et la durée moyenne des phases de repos dépendait du nombre de lactations de la vache. Cette stratégie semble légèrement problématique pour les vaches de deuxième parité, mais potentiellement bénéfique pour les vaches plus âgées., During the transition period in dairy cows, energy demands for milk production are very high, while energy intake is low, leading to a physiologically unavoidable negative energy balance. Physiological imbalance or dysfunction appears to be one of the main factors leading to increased risk for transition diseases, and together, these problems have a great impact on subsequent reproductive performances and culling. This leads to the urgent need for alternative management strategies to help dairy cattle to cope with the intensive systems in which they are raised. Although there has been great improvements in managing the source of energy for the cow, little work has been done in controlling energy expenses to improve energy balance. An incomplete milking in early lactation could help limiting negative energy balance in dairy cattle. The objective of this project was to measure, in a context of commercial dairy farms, the impact of an incomplete milking (without altering the milk frequency) during the first five days in milk (DIM), on performance and health. Specifically, the aims were to quantify its impact on: culling and milk production; on incidence of infectious diseases; on reproductive performance; and on udder sensitivity. A randomized controlled trial was conducted on multiparous cows from a convenient sample of 13 commercial dairy farms. In each herd, all multiparous cows (n = 878) calving between December 2013 and March 2015 were randomly allocated at the time of dry off to a treatment or a control group using a random number generator. Cows in the treatment group were milked incompletely during the first five DIM, with a maximum of 10, 10, 10, 12, and 14 L/d collected on DIM one, two, three, four and five, respectively. Cows in the control group were milked conventionally. Culling hazard and milk yield did not differ among treatment groups. When investigating differences in energy corrected milk yield per week throughout the lactation, incompletely milked cows produced as much as conventionally milked cows during most weeks. Incomplete milking did not affect the odds of new intramammary infection from 11 to 18 DIM, the odds of reproductive tract disease at 35 DIM, or clinical mastitis incidence in the first 90 DIM. The odds of eliminating an existing intramammary infection from 11 to 18 DIM for incompletely milked cows were 2.9 (95% confidence interval: 1.4, 6.0) times those of conventionally milked cows. The incomplete milking protocol had no effect on postpartum luteal activity and it had a positive impact on pregnancy hazard in second parity cows from herds with voluntary waiting period lower than 55 days (180/775 cows). The hazards of pregnancy (95% confidence interval) in incompletely milked cows were 576.3 (240.0, 1383.7), 36.9 (18.9, 72.1), 6.8 (3.3, 13.8), 2.5 (1.0, 5.9), and 0.13 (0.07, 0.26) times that of conventionally milked cows at 1-21, 22-43, 44-65, 66-87 and >87 d after voluntary waiting period, respectively in second parity cows from herds with voluntary waiting period lower than 55 days. The pressure algometer was validated for measuring changes in udder sensitivity due to udder distension. The instrument was shown to be only moderately repeatable for quantifying mechanical nociceptive threshold on the udder and was influenced by extraneous covariates, therefore its use should be considered cautiously or it should be further developed. Consequently, to assess potential increased udder pain due to incomplete milking, we used the resting behavior data of incompletely and conventionally milked animals. There was no effect of incomplete milking on lying time. However, the effect of incomplete milking on frequency of lying bouts and on mean lying bout duration varied by parity level, suggesting that an incomplete milking may be slightly problematic for second parity cows and, possibly, slightly beneficial for older cows.

Staphylococcus aureus is a major pathogen responsible for bovine mastitis, the most common and costly disease affecting dairy cattle. S. aureus naturally releases extracellular vesicles (EVs) during its growth. EVs play an important role in the bacteria-bacteria and bacteria-host interactions and are notably considered as nanocarriers that deliver virulence factors to the host tissues. However, the potential contribution of S. aureus EVs to bacterial pathogenesis has only been explored for human isolates. Moreover, whether EVs play a role in a mastitis context is still unknown. ln this work, we showed that S. aureus Newbould 305 (N305), a bovine mastitis isolate, has the ability to generate EVs in laboratory conditions with a designated protein content including numerous virulence factors. Purified S. aureus N305-secreted EVs were not cytotoxic when tested in vitro on MAC-T and PS, two bovine mammary epithelial cell lines. However, they induced the gene expression of inflammatory cytokines at levels similor to those induced by live S. aureus N305. The in vivo immune response to purified S. aureus N305-secreted EVs was tested in a mouse model for bovine mastitis and their immunogenic effect was compared to that of live S. aureus N305, heat-killed S. aureus N305 and to S. aureus lipoteichoic acid (LTA). Clinical and histopathological signs were evaluated and pro-inflammatory and chemotactic cytokine levels were measured in the mammary gland 24 hour post-inoculation. Live S. aureus induced a significantly stronger inflammatory response thon that of any other condition tested. Nevertheless, S. aureus N305-secreted EVs induced a dose-dependent neutrophil recruitment and the production of a selected set of pro-inflammatory mediators as well as chemokines. This immune response elicited by intramammary S. aureus N305-secreted EVs was comparable to that of heat-killed S. aureus N305 and, partly, by L TA. These results demonstrated that S. aureus N305-secreted EVs induce a mild inflammatory response distinct from the live pathogen, predominantly a chemotaxis related migratory response, after intramammary injection. Overall, our combined in vitro and in vivo data suggest that these EV play a significant role in the pathogenesis of bovine S. aureus mastitis.

Mastitis is an inflammation of the mammary gland associated with worldwide economic losses. Staphylococcus aureus is one of the main etiologic agents of mastitis and triggers several clinical manifestations in the host. Although S. aureus is extensively studied as an agent of nosocomial infections, the mechanisms involved in S. aureus pathogenesis in a mastitis context are not completely understood. Extracellular factors are important components of S. aureus virulence. The production of extracellular vesicles (EVs) by S. aureus has gained attention in the last years. EVs are nano-sized particles formed by the budding of the bacterial membrane. Nowadays, EVs are accepted as a conserved mechanism of secretion that enables inter-kingdom cellular communication. EVs have been purified from different clinical isolates of S. aureus and appear to be a key factor in the infectious process when evaluated using several in vitro and in vivo models. In this thesis, we purified EVs from phylogenetically distant strains of S. aureus isolated from bovine, ovine and human hosts. Ruminant strains were responsible for different degrees of severity of mastitis. S. aureus EVs were strain-dependent in terms of yield, proteins cargo and size. Overall, they were enriched with cytoplasmic and membrane-associated proteins, lipoproteins and virulence factors, including toxins (e.g. PSMs, δ-hemolysin), factors associated to adhesion and colonization (e.g. EbpS, FnBPs), and evasion of host immune system (e.g. Sbi, leukocidin). 44 proteins were shared between all strains, defining a core proteome for S. aureus EVs. We selected the bovine strain S. aureus N305 for further in vitro and in vivo investigations. Although S. aureus N305 EVs were not able to induce cytotoxic effects in bovine mammary epithelial cells (bMEC), they stimulated an immunomodulatory response with an increase of some key inflammatory markers, such as the chemokine IL-8. Purified S. aureus N305 EVs were then tested in a murine model of mastitis. Histopathological results were consistent with the levels of cytokine after 24 h of mammary infection. S. aureus N305 EVs induced a dose-dependent neutrophil recruitment to the alveolar space that can be correlated to the local levels of chemokines. EVs-induced symptoms were although distinct and milder than those induced by the live N305. These results suggest that EVs play a role in S. aureus pathogenesis and can induce an immune response to S. aureus mastitis. Overall, this work opens new perspectives in the development of diagnostic tools and vaccine formulations using native or engineered EVs against S. aureus mastitis.; La mammite est une inflammation de la glande mammaire responsable de pertes lourdes économiques dans le monde. Staphylococcus aureus est l'un des principaux agents étiologiques de la mammite et déclenche plusieurs manifestations cliniques chez l'hôte. Bien que S. aureus soit largement étudié en tant qu'agent d’infections nosocomiales, les mécanismes impliqués dans la pathogenèse de S. aureus dans un contexte mammite sont encore inconnus. Les facteurs extracellulaires sont des composants importants de la virulence de S. aureus. La production de vésicules extracellulaires (VEs) par S. aureus a attiré l'attention au cours de ces dernières années. Les VEs sont des particules de taille nanométrique formées par le bourgeonnement de la membrane bactérienne. De nos jours, les VEs sont considérées comme un mécanisme de sécrétion conservé qui permet la communication cellulaire inter-royaume. Les VEs ont été purifiées à partir de différents isolats cliniques de S. aureus et semblent être un facteur clé dans le processus infectieux lorsqu'ils sont testés en modèles in vitro et in vivo. Dans cette thèse, nous avons purifié des VEs de souches phylogénétiquement distantes de S. aureus, isolées d'hôtes bovins, ovins et humains. Les souches de ruminants étaient responsables de différents degrés de sévérité de mammite. Les caractéristiques de VEs de S. aureus étaient souches-dépendantes en termes de rendement, de contenu protéique et de taille. Elles sont enrichies en protéines cytoplasmiques et membranaires, en lipoprotéines et en facteurs de virulence, notamment en toxines (ex. PSM, δ-hémolysine), en facteurs d'adhésion et de colonisation (ex. EbpS, FnBPs) et d'évasion du système immunitaire de l'hôte (ex. Sbi, leucocidine). 44 protéines sont communes à toutes les souches et définissent un protéome coeur pour les VEs de S. aureus. Nous avons sélectionné la souche bovine S. aureus N305 pour des études fonctionnelles in vitro et in vivo. Bien que les VEs de S. aureus N305 ne soient pas capables d'induire des effets cytotoxiques sur cellules épithéliales mammaires bovines (CEMb), elles induisent une réponse immunomodulatrice avec une augmentation de certains marqueurs inflammatoires clés, tels que la chimiokine IL-8. Des VEs de N305 purifiées ont ensuite été testées dans un modèle murin de mammite. Les résultats histopathologiques étaient en accord avec les taux de cytokines mesurés après 24 h d'infection mammaire. Les VEs de S. aureus N305 ont induit un recrutement de neutrophiles dose-dépendant dans l'espace alvéolaire qui peut être corrélé aux taux locaux de chimiokines. Les symptômes induits par les VEs étaient bien distincts et plus légers que ceux induits par la souches N305 vivante. Ces résultats suggèrent que les VEs jouent un rôle dans la pathogenèse de S. aureus et peuvent induire une réponse immunitaire lors de mammite à S. aureus. Dans l'ensemble, ces travaux ouvrent de nouvelles perspectives dans le développement d'outils diagnostiques et de formulations de vaccins utilisant des VEs naturels ou artificiels contre la mammite à S. aureus.

Escherichia coli are major bacterial pathogens causing bovine mastitis, a disease of great economic impact on dairy production worldwide. This work aimed to study the virulence determinants of mammary pathogenic E. coli (MPEC). By whole-genome sequencing analysis of 40 MPEC and 22 environmental (“dairy-farm” E. coli [DFEC]) strains, we found that only the fec locus (fecIRABCDE) for ferric dicitrate uptake was present in the core genome of MPEC and that it was absent in DFEC genomes (P < 0.05). Expression of the FecA receptor in the outer membrane was shown to be citrate dependent by mass spectrometry. FecA was overexpressed when bacteria were grown in milk. Transcription of the fecA gene and of the inner membrane transport component fecB gene was upregulated in bacteria recovered from experimental intramammary infection. The presence of the fec system was shown to affect the ability of E. coli to grow in milk. While the rate of growth in milk of fec-positive (fec+) DFEC was similar to that of MPEC, it was significantly lower in DFEC lacking fec. Furthermore, deletion of fec reduced the rate of growth in milk of MPEC strain P4, whereas fec-transformed non-mammary gland-pathogenic DFEC strain K71 gained the phenotype of the level of growth in milk observed in MPEC. The role of fec in E. coli intramammary pathogenicity was investigated in vivo in cows, with results showing that an MPEC P4 mutant lacking fec lost its ability to induce mastitis, whereas the fec+ DFEC K71 mutant was able to trigger intramammary inflammation. For the first time, a single molecular locus was shown to be crucial in MPEC pathogenicity., IMPORTANCE Bovine mastitis is the major infectious disease in dairy cows and the leading cause of economic loss to the global dairy industry, directly contributing to the price of dairy products on supermarket shelves and the financial hardships suffered by dairy farmers. Mastitis is also the leading reason for the use of antibiotics in dairy farms. Good farm management practices in many countries have dramatically reduced the incidence of contagious mastitis; however, the problems associated with the incidence of environmental mastitis caused by bacteria such as Escherichia coli have proven intractable. E. coli bacteria cause acute mastitis, which affects the health and welfare of cows and in extreme cases may be fatal. Here we show for the first time that the pathogenicity of E. coli causing mastitis in cows is highly dependent on the fecIRABCDE ferric citrate uptake system that allows the bacterium to capture iron from citrate. The Fec system is highly expressed during infection in the bovine udder and is ubiquitous in and necessary for the E. coli bacteria that cause mammary infections in cattle. These results have far-reaching implications, raising the possibility that mastitis may be controllable by targeting this system.

Staphylococcus aureus is the major cause of very severe mastitis of dairy goats. The initial objective of our study was to fine-tune an experimental model of infection of the goat mammary gland with two strains of S. aureus and two lines of goats (low and high somatic cell score lines). Following the challenge, the 10 infected goats divided in two clear-cut severity groups, independently of the S. aureus strain and the goat line. Five goats developed very severe mastitis (of which four were gangrenous) characterized by uncontrolled infection (UI group), whereas the other five kept the infection under control (CI group). The outcome of the infection was determined by 18 h post-infection (hpi), as heralded by the bacterial milk concentration at 18 hpi: more than 107/mL in the UI group, about 106/mL in the CI group. Leukocyte recruitment and composition did not differ between the groups, but the phagocytic killing at 18 hpi efficiency did. Contributing factors involved milk concentrations of α-toxin and LukMF' leukotoxin, but not early expression of the genes encoding the pentraxin PTX3, the cytokines IL-1α and IL-1β, and the chemokines IL-8 and CCL5. Concentrations of TNF-α, IFN-γ, IL-17A, and IL-22 rose sharply in the milk of UI goats when infection was out of control. The results indicate that defenses mobilized by the mammary gland at an early stage of infection were essential to prevent staphylococci from reaching critical concentrations. Staphylococcal exotoxin production appeared to be a consequent event inducing the evolution to gangrenous mastitis.

En France, plusieurs facteurs incitent actuellement les éleveurs à modifier leurs pratiques de gestion de la santé animale. Les plans d'action EcoAntibio mis en place par le ministère de l'agriculture visent une diminution de l'utilisation des antibiotiques dans le secteur de l'élevage. En agriculture biologique, le cahier des charges limite l'utilisation de traitements vétérinaires conventionnels et sollicite les éleveurs à utiliser des traitements curatifs alternatifs. Les éleveurs conventionnels et biologiques s'intéressent donc de plus en plus à des médecines dites "alternatives". Parmi elles, l'aromathérapie se base sur l'utilisation des huiles essentielles pour leurs propriétés thérapeutiques. Dans les élevages français, cette pratique semble relativement développée dans certains secteurs, notamment en élevages bovins laitiers. Cette utilisation est d'autant plus marquée dans les élevages biologiques. Dans ces élevages laitiers, l'utilisation de l'aromathérapie se fait principalement dans le cas des mammites. L'efficacité de cette pratique sur les mammites n'est pas scientifiquement prouvée. Quelques essais ont été menés en collaboration avec des éleveurs pour essayer de mettre en évidence cette efficacité. Les résultats sont mitigés, avec une guérison clinique satisfaisante mais une guérison bactériologique peu concluante. Outre le manque de preuves en termes d'efficacité, l'aromathérapie est confrontée à un autre point de blocage : la règlementation. En élevage, l'utilisation thérapeutique de certaines substances végétales, dont un grand nombre d'huiles essentielles, n'entre pas dans le cadre de la médecine vétérinaire légale., In France, several factors currently encourage breeders to modify their animal health management practices. The EcoAntibio Action Plans launched by the Ministry of Agriculture aim to reduce use of antibiotics in livestock. In organic farming, the specification limits use of conventional veterinary treatments and ask breeders to use alternative curative treatments. Conventional and organic breeders are increasingly interested in so-called "alternative" medicines. Among them, aromatherapy is based on use of essential oils for their therapeutic properties. In French farms, this practice has developed in some sectors, particulary dairy cattle farms. This use is even more marked in organic farms. In these dairy farms, aromatherapy is mainly use for mastitis treatment. The effectiveness of this practice on mastitis is not scientifically proven. Some trials have been conduced in collaboration with breeders to highlight this effectiveness. The results are lukewarm, with a clinical cure but an inconclusive bacteriological cure. In addition to the lack of evidence in terms of efficiency, aromatherapy is facing antoher blocking point : regulation. In the breeding, the therapeutic use of certain substances, including many essential oils, does not fall within the scope of legal veterinary medicine.

National audience; The concentration of milk cells, or Somatic Cell Count (SCC), is an indirect indicator of mammary infection, which is widely used in the epidemiology of mastitis. It is also a predictor of intra-mammary infection susceptibility used for selection of dairy ruminants resistant to mastitis. Owing to the role attributed to milk cells in the immune protection against mastitis, the selection of animals with very low SCC has given rise to doubts and criticism, fearing that selection would lead to an increased susceptibility to mastitis. Epidemiological surveys have yielded apparently contradictory results, which did not settle this issue. Nevertheless, by taking into account the immunobiology of the mammary gland, along with the results of recent studies on SCC-based divergent selection of dairy animals, it is possible to give a definite answer to the question "Should we avoid selecting dairy animals with very low SCC?" It clearly appears that selection does not reduce the baseline SCC of uninfected healthy glands and does not weaken the capacity of the mammary gland to defend itself against infections.; La concentration en cellules du lait (CCS) est un indicateur indirect d’une infection mammaire très utilisé en épidémiologie, mais aussi un prédicteur de la sensibilité aux infections mammaires utile pour la sélection génétique d’animaux plus résistants aux mammites. En raison du rôle de protection immunitaire attribué aux cellules du lait, des réserves sont émises à l’encontre de la sélection d’animaux dont le lait serait pauvre en cellules, ce qui entrainerait une sensibilité accrue aux mammites. Des enquêtes épidémiologiques apparemment contradictoires n’ont pas permis de lever ces réticences. Cependant, une analyse critique des connaissances sur l’immunité mammaire et des études récentes réalisées sur des animaux issus d’une sélection divergente sur les valeurs génétiques du caractère « Cellules » permettent de répondre à la question « Faut-il éviter de sélectionner des animaux laitiers dont les CCS sont très faibles ? ». Il apparaît clairement qu’en l’absence d’infection, les valeurs de CCS sont physiologiquement très faibles et ne sont pas modifiées par la sélection, qui ne réduit pas non plus la capacité de la mamelle à se défendre contre les infections.

Le logiciel Herd Navigator™ est un outil d’élevage de précision effectuant à intervalles réguliers des mesures de l’activité de la LDH dans le lait. L’objectif de cette étude était de déterminer l’exactitude des alarmes mammite déclenchées par ce logiciel. Au sein d’un élevage, les 295 vaches ayant déclenché cette alarme ou dont l’activité de la LDH dépassait 40 U/L ont fait l’objet de deux examens cliniques à quelques jours d’intervalle (CMT, température rectale, examen vaginal, score de boiterie). L’alarme mammite présente ainsi une valeur prédictive positive de 82.35% dès le premier examen. Plus de la moitié des mammites (55,4%) sont subcliniques. De plus, une activité de la LDH supérieure à 40 U/L, même en-dehors d’une situation d’alarme, est révélatrice d’une mammite dans 63.75% des cas (100% des cas si activité supérieure à 100 U/L). L’éleveur doit donc examiner et si besoin traiter les vaches en alarme ou ayant une activité de LDH élevée.

International audience; The urge to reduce antimicrobials use in dairy farming has prompted a search for alternative solutions. As infections of the mammary gland is a major reason for antibiotic administration to dairy ruminants, mammary probiotics have recently been presented as a possible alternative for the treatment of mastitis. To assess the validity of this proposal, we performed a general appraisal of the knowledge related to probiotics for mammary health by examining their potential modes of action and assessing the compatibility of these mechanisms with the immunobiology of mammary gland infections. Then we analyzed the literature published on the subject, taking into account the preliminary in vitro experiments and the in vivo trials. Preliminary experiments aimed essentially at exploring in vitro the capacity of putative probiotics, mainly lactic acid bacteria (LABs), to interfere with mastitis-associated bacteria or to interact with mammary epithelial cells. A few studies used LABs selected on the basis of bacteriocin production or the capacity to adhere to epithelial cells to perform in vivo experiments. Intramammary infusion of LABs showed that LABs are pro-inflammatory for the mammary gland, inducing an intense influx of neutrophils into milk during lactation and at drying-off. Yet, their capacity to cure mastitis remains to be established. A few preliminary studies tackle the possibility of using probiotics to interfere with the teat apex microbiota or to prevent the colonization of the teat canal by pathogenic bacteria. From the analysis of the published literature, it appears that currently there is no sound scientific foundation for the use of probiotics to prevent or treat mastitis. We conclude that the prospects for oral probiotics are not promising for ruminants, those for intramammary probiotics should be considered with caution, but that teat apex probiotics deserve further research.