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Intramammary infections and mastitis are a leading cause of diseases, death and production losses in dairy ruminants. Increased susceptibility to mastitis during the peripartal period has been yet widely documented. Negative energy balance is frequent in early lactation and considered as a known risk factor for mastitis. The relationship between energy balance and mastitis susceptibility however remained unclear and poorly described. The aim of my thesis was to examine the relation between a negative energy balance and susceptibility to mastitis in dairy ewes. In particular, my research works showed an enhanced susceptibility to energy restriction in mastitis-susceptible ewes., Les infections intra-mammaires (IMI) responsables de mammites, sont une cause importante de maladie, de mortalité et de pertes de production chez les ruminants laitiers. Le péri-partum est une période où la fréquence des mammites est significativement plus élevée. Parmi les facteurs de risques, le déficit énergétique, fréquent au début de la lactation, est souvent évoqué. Cependant, les bases mécanistiques de l’éventuelle relation entre IMIs et déficit énergétique sont encore peu comprises. L’objectif de ma thèse était ainsi d’évaluer la relation éventuelle entre le déficit énergétique et la prédisposition aux mammites en utilisant la brebis laitière comme modèle. Mes travaux ont montré que les brebis génétiquement prédisposées aux mammites ont également une sensibilité accrue au déficit énergétique et à la cétose.

Individual risk factors for clinical mastitis within the first month of lactation (early clinical mastitis, ECM) were analyzed in 695 Holstein multiparous cows (561 ECM- and 134 ECM+) which were clinical mastitis-free during the previous lactation. They belonged to 44 herds from the French Brittany region and were surveyed for 4 years. Individual biological parameter levels during the last 2 months of gestation, production of the preceding lactation and calving conditions and health were the available explanatory variables. The construction and explanation model used multiple logistic regression run with herd as a fixed effect. High precalving plasma ceruloplasmin oxidase (p < 0.01) and low glutamate dehydrogenase (p < 0.05) activities, high 305-day previous-lactation milk yield (p < 0.001) and milk protein content at the last milk-test day before drying-off (p < 0.01) were the significant risk factors for ECM occurrence. Intensive production and unsatisfactory dietary conditions (energy and antioxidants) would increase ECM risk in the dairy cow. Further studies should be conducted to determine if metabolic and/or genetic factors could explain the relationship between ceruloplasmin and subsequent ECM occurrence.

Staphylococcus aureus is an opportunistic pathogen of humans and warm-blooded animals. It is foundin healthy carriage and can cause a wide range of infections in its hosts. The epithelial surface is theprincipal site of staphylococcal infection. The pathogens have evolved sophisticated mechanisms tohijack the main function of the host cells, thus promoting their invasion and colonization. Amongstthese effects are, alteration of host cell apoptosis, promotion of cell proliferation or conversely,inhibition of cell growth. Using human cervix cancer HeLa and mammary gland bovine MAC-Tepithelial cells, we studied the cytopathic effect of S. aureus and its ability to affect host cell cycle.Using microscopic examination, we found that S. aureus induced the cytopathic effect resulting in theenlargement of the host cells 72 hours post infection. Our experiments showed that the proliferationrate of the epithelial cells was slowed down in S. aureus-infected culture. Three different S. aureusstrains from human and animal origins were used and showed similar effect on both human and bovinecells. Microscopic analysis of DAPI-stained cells revealed huge decrease of mitotic indexes in infectedHeLa and Mac-T cells, synchronized by Double Thymidine Block. Using flow cytometry analysis, wefound that S. aureus induces G2/M phase delay in synchronous HeLa cells. By Western blot analysis,we showed that the G2/M phase delay was associated with accumulation of inactive phosphorylatedcyclin-dependent kinase Cdk1/cdc2, a key inducer of mitosis entry, and dephosphorylation of corehistone H3, an important component of the epigenetic landscape.These results suggest that the delay in G2/M phase transition of the infected epithelial cells may beone of the mechanisms employed by S. aureus to survive and to propagate inside the host. A fullcomprehension of the mechanisms associated to S. aureus-host cell interactions will undoubtedly helpto discover novel targets to treat staphylococcal infections.

Staphylococcus aureus is a major causative agent of intramammary infections in dairy cows. In this report, the pathogenesis of these infections is described. The potential role in virulence of S. aureus surface components (adhesins, protein A and capsular polysaccharides), toxins, extracellular enzymes and coagulase, and perspectives for the development of an efficient vaccine are discussed.

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National audience; The last thirty years have seen an important increase of cattle diseases, especially mastitis, which is the inflammation of the mammary gland. Mastitis is nowadays considered as the first source of financial impairments within nursing and dairy cattle in France and Europe. Vaccines or therapy development requires an accurate knowledge of the mammary response during mastitis. Mammary epithelial cells (MECs), by their potential of pathogens recognition and secretion of pro-inflammatory cytokines and chemokines, likely act as "sentinels" for the triggering of the immune response. Nevertheless, precise mechanisms ofthis role remain to be deciphered. Recent results suggest the implication of IL-17, an interleukin of the IL-23/IL-17 axis, which is important for bacterial clearance in human and mice. In addition, studies of the response induced by Escherichia coli essentially focus on the P4 strain, although this response possibly varies among the E. coli strains. This study aims at characterizing the outlines of the response induced by four strains of E. coli isolated from mastitis by, first, confirming the protective role of MECs in the triggering of the inflammation and the reinforcement of leukocytes action. In a second part, by defining the implication of IL-17 and IL-22 and determining the key molecules of defense and leukocytes recruitment. Finally, by characterizing the chronology of the immune response. This study shows that a synergy between IL-17 and E. coli would increase the Immune response of MECs, and the outline of immune response varies depending on E. coli strain.

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Hors-série "Parasitologie des ruminants laitiers"; National audience; Les champignons sont responsables de mammites dans 1 à 6 % des cas chez la vache. La majorité des mammites mycosiques survient à la suite d’un traitement antibiotique intra-mammaire. Les protothèques sont des algues microscopiques dont l’importance comme agents de mammites est sous-estimée. La pénétration des agents fongiques ou des protothèques s’effectue essentiellement par voie diathélique. Les facteurs favorisants de ces mammites sont les mauvaises conditions d’hygiène et les traitements intra-mammaires. L’infection reste le plus souvent subclinique. Seul un examen microbiologique permet d’établir le diagnostic de certitude. Le traitement des affections fongiques repose sur l’arrêt de l’antibiothérapie et sur la vidange fréquente et complète du quartier malade. La prévention des mammites mycosiques ou à protothèques inclut la surveillance de l’hygiène des locaux, du personnel, du matériel de traite, de la mamelle et des trayons, l’isolement des animaux atteints et l’élimination des animaux qui souffrent de mammites mycosiques graves ou chroniques.

The objective of the study was to identify the risk factors for clinical mastitis and for new subclinical intramammary infection in dairy heifers around calving. 855 heifers from 57 French dairy herds were included. Only clinical mastitis occurring from 3 weeks before to 4 weeks after calving were considered. New intramammary infections were determined based on 2 quarter milk samples, implemented 2 days before and 15 days after calving. 16 and 8 risk factors were identified for clinical mastitis and for new intramammary infection respectively. They were related to cow individual traits (housing with mature cows before calving, dystocia, udder oedema, high milk production level, low milk urea…) and to herd context and management (high incidence rate of clinical mastitis, small sized lying area, overmilking,…).; Cette étude avait pour objectif d’évaluer les facteurs de risque de mammite clinique et de nouvelle infection à expression subclinique chez les vaches primipares au vêlage. 855 vaches primipares issues de 57 élevages laitiers de Bretagne et Pays de la Loire ont été incluses dans l’enquête. Seules les mammites cliniques survenant entre 3 semaines avant à 4 semaines après vêlage ont été étudiées. Les nouvelles infections ont été déterminées sur la base de 2 prélèvements de lait de quartier, 2 jours avant et 15 jours après vêlage. 16 et 8 facteurs de risque ont été identifiés pour les mammites cliniques et les nouvelles infections respectivement. Ils étaient relatifs aux caractéristiques individuelles des vaches (logement antepartum non séparé des adultes, conditions de vêlage difficiles, œdème mammaire, niveau de production laitière élevé, taux d’urée faible...) ainsi qu’au contexte de troupeau (fréquence élevée de mammites cliniques, surface d’aire paillée insuffisante, pratique de la surtraite,...).

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International audience; Le programme à visée nationale "Objectif Zéro Mammite" (ZM), conduit dans 534 élevages adhérents du Contrôle Laitier représentatifs des élevages français du point de vue des races et de la localisation géographique, a eu pour objectif de mettre en évidence les pratiques reliées au contrôle optimum des infections mammaires dans les conditions françaises d’élevage. Deux groupes d’élevages ont été sélectionnés et comparés sur la base des scores cellulaires annuels d’élevage (SCS) calculés à partir des comptages individuels mensuels. L’un comprend 326 élevages parmi les plus bas SCS au niveau national et l’autre 208 élevages parmi les plus proches du SCS médian, leurs SCS moyens correspondant à des comptages moyens de 125 000 cellules / ml et 270 000 cellules / ml respectivement. Dans ces élevages, des données concernant exploitations, troupeaux, animaux, logement, pratiques alimentaires, hygiène, prévention des mammites, tarissement et traite ont été recueillies par questionnement des éleveurs et visite de traite. Outre l’effectif de vaches traites, les pratiques d’hygiène de traite qui ont caractérisé les élevages à SCS très bas ont été : la pulvérisation du produit de post-trempage sur les trayons, le changement de lavettes entre 2 traites, le rinçage et la désinfection des griffes après passage d’une vache infectée, la pratique du CMT avant la traite pour les vaches douteuses et un trayeur travaillant avec précision. A l’inverse, traire les vaches à cellules élevées avec un faisceau spécial, nettoyer les seuls trayons sales avant la traite, pratiquer le pré trempage en utilisant un produit moussant et le post trempage avec un gobelet anti-retour sont des pratiques associées aux élevages à SCS moyens.

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The effect of mastitis and related-germs on milk chemical composition (protein and lac- tose contents) and milk somatic cell count (SCC) was investigated in 501 milk quarter samples during two consecutive years in cows from three experimental herds. Each infected quarter was matched by a healthy one in the same udder, as a control. Milk protein and mineral assays were performed in a sub- sample of 128 milks. Staphylococci were the most frequently isolated germs (Staphylococcus aureus: 27%, coagulase-negative Staphylococci: 26%, Streptococci: 21%). Major milk pathogens (Staphylococcus aureus, Streptococcus uberis or Escherichia coli) associated with clinical signs of mastitis were accompanied by higher SCC (+1.6 log·mL -1 , P < 0.01), lower lactose concentration (-7.6 g·kg -1 , P < 0.01), higher protein concentration (+3.3 g·kg -1 , P < 0.01) and higher soluble protein concentrations (IgG and BSA), hence a sharp decrease in the casein/protein ratio (-10 percentage points, P < 0.01). Changes were more marked when Escherichia coli was present. Corynebacterium bovis did not alter milk chemical composition whereas coagulase-negative Staphylococci slightly re- duced lactose concentration (-1.8 g·kg -1 ) and increased SCC (+0.37 log·mL -1 ). Calcium and phos- phorus milk contents were hardly modified by the presence of microorganisms. The decrease in milk yield during clinical mastitis varied from 1.6 kg·d -1 in the presence of Staphylococcus aureus to 15 kg·d -1 in the presence of Escherichia coli.

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no. spécial: Le bulletin d'Epiter

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The relationships between the herd-level distribution of udder health measures (traumatic, physical, functional and congestive udder disorders, clinical mastitis and somatic cell counts (SCC) greater than 400 x 10(3) ml-1) and farm (general hygiene, milking practices and housing system) and aggregated individual characteristics were studied by canonical correspondence analysis (CCA). This is a correspondence-analysis technique in which the axes representing disease patterns are expressed as linear combinations of potentially explanatory variables. These main axes can be drawn so that the distribution of diseases for combinations of the main-explanatory managerial variables can be visualized. The type of quarantine for newcomers, type of animal housing, presence of shelter at pasture, use of udder towel before milking, milk production and loss of body condition after calving, were strongly correlated with udder health status. Mastitis and physical udder disorders were associated with the combination of no introduction of replacement heifers and low loss of body condition after calving. Congestive disorders were more common on farms when no quarantine for newcomers was combined with high loss of body condition after calving. Higher clinical and subclinical mastitis incidence risks were associated with the combination of no udder towel, loose housing with high animal density and high milk production. In most cases, clinical mastitis and high SCC were explained by the same covariates but with contrasting levels of those covariates.

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Associations among diseases in dairy cows were studied on a two-consecutive-lactation basis by means of a longitudinal and prospective study carried out on 24 French herds. Associations between diseases of a given lactation and diseases of the previous lactation were adjusted for breed, parity, milk yield, season of calving, calving interval and type of housing, using multiple logistic regression models with random effects. Five of the 12 associations highlighted are recurrences (clinical mastitis within 5 days after calving, placental retention, lameness other than interdigital pododermatitis, reproductive disorders, milk fever). Otherwise, the study confirms the main role of placental retention in associations among diseases.

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Bovine milk is generally considered to be almost devoid of complement, on the basis of undetectable hemolytic activity, unless inflammation recruits plasma components. This study examines the deposition of complement components on a mastitis-causing isolate of Streptococcus agalactiae by enzyme-linked immunosorbent assay (ELISA). Neat milk from mid-lactating, uninflamed mammary glands (normal milk) effected marked C3 deposition on bacteria. Kinetic studies showed a protracted lag period (30 to 45 min) preceding C3 deposition, which required about 2 h to reach a maximum. Experiments with diluted serum suggested that this slow C3 deposition resulted mainly from the low concentration of certain components of complement in milk. Bacteria incubated in neat milk readily bound bovine conglutinin, indicating the presence of iC3b. Elution of covalently bound C3 fragments with hydroxylamine confirmed the deposition of C3b and iC3b on bacteria. Deposition of C4 on bacteria was not detected in neat milk, suggesting that C3 deposition did not result from the activation of the classical pathway. This was not the result of a lack of antibodies. However, C4 deposition could be obtained by adding purified bovine C1q to normal milk, and C3 deposition was accelerated, suggesting the participation of the classical pathway. The deposition of C1q on antibody-sensitized bacteria was impeded by milk compared with that of C1q diluted in phosphate-buffered saline. Concentrations of C1q in normal milk were very low, ranging from 150 to 250 ng/ml. Overall, these findings indicate that C1q was a limiting factor of the classical pathway in normal milk. The capacity of milk to deposit C3 fragments on mastitis-causing S. agalactiae prompts further studies to investigate its role in opsonophagocytosis.

Experimental mastitis induced with a single intramammary injection of Listeria monocytogenes was compared with two naturally occurring cases. Four strains of L. monocytogenes, two of serotype 1/2a and two of serotype 4b were used for the experimental infections and two diametrically opposed quarters of four cows were inoculated with 300 cfu. Bacteriological examination and somatic cell counts of quarter foremilk samples were performed weekly for at least 6 months after challenge. All the inoculated quarters developed chronic subclinical mastitis with occasional clinical episodes. The results were similar to those observed in natural listeria mastitis. Four experimentally infected quarters were treated during lactation (gentamicin and cloxacillin) or at "drying-off" (cloxacillin), or at both times. Only one of four quarters was cured after treatment only at "drying-off". All experimental and naturally infected animals were slaughtered and bacteriological examination was performed on liver, spleen and supramammary, iliac and mesenteric lymph nodes. L. monocytogenes strains were isolated from the supramammary lymph nodes of two experimentally and two naturally infected cows and from an iliac lymph node from one of the naturally infected cows. The epidemiological data were supported by serotyping, lysotyping and DNA macro-restriction analysis. The experimental model of listeria mastitis mimics spontaneous cases and should be useful in further studies of listeria mastitis.

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This study examined the role of antibodies against the X-protein, a surface-localized antigen frequently associated with streptococci causing mastitis in cattle, in the opsonization and phagocytosis of unencapsulated Streptococcus agalactiae . The analysis of various strains of serotype NT/X by flow cytometry, after labeling with a monoclonal antibody to X-protein, revealed that they consisted of a mixture of unstained and stained bacteria. Cloning of mother strains yielded clones of unstained bacteria but not homogeneous clones of stained bacteria. Analysis by ELISA of an unstained clone (4.1) derived from the reference NT/X strain 24/60 indicated that it expressed low amount of X-protein at its surface, about 25 times less than the stained clone 24/60 5.6. Colloidal gold immunolabeling showed the X-protein at the periphery of bacteria (of clone 5.6 and in lower amount of clone 4.1), at a distance from the cell wall. Bovine antibodies (essentially IgG) to X-protein behaved like the monoclonal antibody in the cytometric assay. They activated the classical pathway of complement as shown by the deposition of C1q and C4 on bacteria. Deposition of C4 also occurred on the low-surface-producing clone 4.1 in the presence of antibodies to X-protein, although less efficiently than on the high-surface-producing clone 5.6. When used alone, antibodies promoted the ingestion of bacteria and heat-inactivated immune serum promoted the chemiluminescence activity and the killing by polymorphonuclear cells. In conclusion, antibodies to X-protein induced the deposition of C3 by the classical pathway and were also able to stimulate opsonophagocytic killing of X-bearing S. agalactiae in the absence of deposited C3.

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chap. 3; National audience