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International audience; Infections of the mammary gland remain a frequent disease of dairy ruminants that negatively affect animal welfare, milk quality, farmer serenity, and farming profitability and cause an increase in use of antimicrobials. There is a need for efficacious vaccines to alleviate the burden of mastitis in dairy farming, but this need has not been satisfactorily fulfilled despite decades of research. A careful appraisal of past and current research on mastitis vaccines reveals the peculiarities but also the commonalities among mammary gland infections associated with the major mastitis pathogens Escherichia coli, Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae, or Streptococcus dysgalactiae. A major pitfall is that the immune mechanisms of effective protection have not been fully identified. Until now, vaccine development has been directed toward the generation of antibodies. In this review, we drew up an inventory of the main approaches used to design vaccines that aim at the major pathogens for the mammary gland, and we critically appraised the current and tentative vaccines. In particular, we sought to relate efficacy to vaccine-induced defense mechanisms to shed light on some possible reasons for current vaccine shortcomings. Based on the lessons learned from past attempts and the recent results of current research, the design of effective vaccines may take a new turn in the years to come.

Neutrophils that reside in the bone marrow are switly recruited from circulating blood to fight infections. For a long time, these first line defenders were considered as microbe killers. However their role is far more complex as cross talk with T cells or dendritic cells have been described for human or mouse neutrophils. In cattle, these new roles are not documented yet. We identified a new subset of regulatory neutrophils that is present in the mouse bone marrow or circulate in cattle blood under steady state conditions. These regulatory neutrophils that display MHC-II on the surface are morphologically indistinguishable from classical MHC-IInegneutrophils. However MHC-IIposand MHC-IInegneutrophils display distinct transcriptomic profiles. While MHC-IInegand MHC-IIposneutrophils display similar bacterial phagocytosis or killing activity, MHC-IIposonly are able to suppress T cell proliferation under contact-dependent mechanisms. Regulatory neutrophils are highly enriched in lymphoid organs as compared to their MHC-IInegcounterparts and in the mouse they express PDL-1, an immune checkpoint involved in T-cell blockade. Our results emphasize neutrophils as true partners of the adaptive immune response, including in domestic species. They open the way for discovery of new biomarkers and therapeutic interventions to better control cattle diseases.

International audience

Type 3 immunity encompasses innate and adaptive immune responses mediated by cells that produce the signature cytokines IL-17A and IL-17F. This class of effector immunity is particularly adept at controlling infections by pyogenic extracellular bacteria at epithelial barriers. Since mastitis results from infections by bacteria such as streptococci, staphylococci and coliform bacteria that cause neutrophilic inflammation, type 3 immunity can be expected to be mobilized at the mammary gland. In effect, the main defenses of this organ are provided by epithelial cells and neutrophils, which are the main terminal effectors of type 3 immunity. In addition to theoretical grounds, there is observational and experimental evidence that supports a role for type 3 immunity in the mammary gland, such as the production of IL-17A, IL-17F, and IL-22 in milk and mammary tissue during infection, although their respective sources remain to be fully identified. Moreover, mouse mastitis models have shown a positive effect of IL-17A on the course of mastitis. A lot remains to be uncovered before we can safely harness type 3 immunity to reinforce mammary gland defenses through innate immune training or vaccination. However, this is a promising way to find new means of improving mammary gland defenses against infection.

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.

International audience; An enduring controversy exists about low milk cell counts and susceptibility to mastitis. The concentration of milk leukocytes, or somatic cell count (SCC), is a well-established direct indicator of mammary gland inflammation that is highly correlated with the presence of a mammary infection. The SCC is also used as a trait for the selection of dairy ruminants less prone to mastitis. As selection programs favor animals with less SCC, and as milk cells contribute to the defense of the mammary gland, the idea that susceptibility to mastitis could possibly be increased in the long term has been put forward and is still widely debated. Epidemiological and experimental studies aimed at relating SCC to susceptibility to mastitis have yielded results that seem contradictory at first sight. Nevertheless, by taking into account the immunobiology of milk and mammary tissue cells and their role in the defense against infection, along with recent studies on SCC-based divergent selection of animals, the issue can be settled. Apparent SCC-linked susceptibility to mastitis is a phenotypic trait that may be linked to immunomodulation but not to selection.

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.

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.

Ruminants are the main source of human infections with the obligate intracellular bacterium Coxiella (C.) burnetii. Infected animals shed high numbers of C. burnetii by milk, feces, and birth products. In goats, shedding by the latter route coincides with C. burnetii replication in epithelial (trophoblast) cells of the placenta, which led us to hypothesize that epithelial cells are generally implicated in replication and shedding of C. burnetii. We therefore aimed at analyzing the interactions of C. burnetii with epithelial cells of the bovine host (1) at the entry site (lung epithelium) which govern host immune responses and (2) in epithelial cells of gut, udder and placenta decisive for the quantity of pathogen excretion. Epithelial cell lines [PS (udder), FKD-R 971 (small intestine), BCEC (maternal placenta), F3 (fetal placenta), BEL-26 (lung)] were inoculated with C. burnetii strains Nine Mile I (NMI) and NMII at different cultivation conditions. The cell lines exhibited different permissiveness for C. burnetii. While maintaining cell viability, udder cells allowed the highest replication rates with formation of large cell-filling Coxiella containing vacuoles. Intestinal cells showed an enhanced susceptibility to invasion but supported C. burnetii replication only at intermediate levels. Lung and placental cells also internalized the bacteria but in strikingly smaller numbers. In any of the epithelial cells, both Coxiella strains failed to trigger a substantial IL-1β, IL-6 and TNF-α response. Epithelial cells, with mammary epithelial cells in particular, may therefore serve as a niche for C. burnetii replication in vivo without alerting the host's immune response.

Critical appraisal of vaccine approaches to control bovine mastitis. Despite their interest to combat mastitis, vaccines are few and underused, owing to limited efficacy. The mammary gland may have established with its pathogens a modus vivendi that favours the settlement of chronic subclinical infections. Reprogramming this immune response in a way that favours bacteriological cure but spares the secretory function presents a major challenge to immunologists. To improve vaccine efficacy, stress should be put on research on T lymphocyte-dependent immune defenses. In this view, improved knowledge of mammary lymphocyte populations, in association with systems vaccinology approaches, should offer new prospects for the development of efficient vaccines., Les vaccins contre les mammites sont peu nombreux à être commercialisés et peu utilisés en raison d’une efficacité limitée. Il semble que la mamelle ait établi avec ses agents pathogènes un modus vivendi qui favorise le maintien d’infections chroniques subcliniques. La reprogrammation de cette immunité par la vaccination dans un sens favorisant l’élimination des infections sans compromettre la fonction sécrétoire pose un défi majeur aux immunologistes. Pour améliorer l’efficacité des vaccins, les défenses immunitaires dépendantes des lymphocytes T ne devraient plus être négligées. Dans cette optique, une meilleure connaissance des populations lymphocytaires mammaires, associée aux approches de la vaccinologie systématique, devrait offrir de nouvelles perspectives de développement de vaccins efficaces., Rainard Pascal. Examen critique des approches vaccinales pour lutter contre les mammites bovines. In: Bulletin de l'Académie Vétérinaire de France tome 168 n°1, 2015. pp. 61-66.

International audience; Bovine mastitis is one of the most common diseases in the dairy industry and it is a major welfare problem. Pain during mastitis is generally assessed through behavior but a combination of indicators would increase the chances of detecting pain and assessing its intensity. The aim of this study was to assess behavioral and patho-physiological responses as possible signs of pain experienced by cows after experimental intramammary challenge (mastitis) with Escherichia coli. Six Holstein Friesian cows received an inoculation of E.coli P4 in one healthy quarter. Evolution of the disease was assessed using bacteriological growth and somatic cell counts (SCC). Cows' response to the challenge was monitored by direct behavioral and clinical observations, data loggers, rumen temperature sensors, and indicators of inflammation, stress, and oxidative status. From all data recorded, the variables that contributed most to the discrimination of mastitis phases were obtained by factorial discriminant analysis. Baseline levels of all indicators corresponded to values before challenge. Specifically, we weighted data relating to lying behavior by the observations at the same hour of the day before challenge to eliminate the circadian rhythm effect. We identified 3 phases that were discriminated by factorial discriminant analysis with good performance. Nine indicators varied according to the phase of the disease: cows' attitude toward their surroundings, tail position, clinical signs, ear position, variation of postural changes, concentrations of haptoglobin and serum amyloid A (SAA), cortisol blood levels, and rumen temperature (as a surrogate for body temperature). In phase 1 (4 to 8 h postinoculation), E. coli proliferated exponentially in milk but inflammation indicators remained at baseline levels. Cows were less attentive toward their surroundings (median score, 0.63), and postural changes (lying/standing) were less frequent (0.75 times from baseline). In phase 2 (12 to 24 h postinoculation), bacterial concentrations peaked around 12 h and then began to decrease concomitantly with a sharp SCC increase. Cows were less attentive toward their surroundings (score, 0.54), had high plasma cortisol (31.3 ng/mL) and SAA (100.3 mu g/mL) concentrations, and rumen temperature was increased (40.3 degrees C). In phase 3 (32 to 80 h postinoculation), bacterial concentrations decreased concomitantly with high SCC levels. Cows had high levels of haptoglobin (0.57 mg/mL) and SAA (269 mu g/mL) but showed no behavioral changes. Dairy cows displayed changes of behavioral, inflammatory, and stress parameters after E. coli mammary inoculation. Our results suggest that cows may have experienced discomfort in the preclinical phase (phase 1) and pain in the acute phase (phase 2) but neither discomfort nor pain in the remission phase (phase 3). Although larger controlled studies are needed to confirm our findings, this knowledge could be useful for early detection of E. coli mastitis and for decision-making regarding the initiation of pain-relief treatment during mastitis in dairy cows. This would improve animal welfare and potentially faster disease remission.

International audience; The distal compartments of the udder are the first to interact with invading pathogens. The regulatory and effector functions of two major teat regions [Fürstenberg's rosette (FR); teat cistern (TC)] are largely unknown. The objective of this study was to establish an in vitro model with explants of the FR and the TC to analyse their response towards Escherichia coli LPS and Staphylococcus aureus lipoteichoic acid (LTA). Quantitative stereological analysis confirmed differences in the cellular composition of FR and TC explants. Chemokine (CXCL8, CCL5, CCL20) and TNF-α mRNA were expressed at low levels in both locations. Explant stimulation with LPS increased the mRNA abundance of all tested chemokines and TNF-α. Stimulation with LTA only induced CCL20 and CXCL8. LPS- and LTA-stimulated explant supernatants contained CXCL8 and CXCL3. Supernatants significantly attracted neutrophils in vitro. Compared with TC, the FR showed high constitutive mRNA expression of S100 proteins (A8, A9, A12). In the TC, both LPS and LTA significantly induced S100A8, whereas S100A9 and S100A12 expression was only induced by LPS. The novel model system underpins the role of the teat for recognising pathogens and shaping a pathogen- and location-specific immune response.

The cytokine IL-17A has been shown to play critical roles in host defense against bacterial and fungal infections at different epithelial sites, but its role in the defense of the mammary gland (MG) has seldom been investigated, although infections of the MG constitute the main pathology afflicting dairy cows. In this study, we showed that IL-17A contributes to the defense of the MG against Escherichia coli infection by using a mouse mastitis model. After inoculation of the MG with a mastitis-causing E. coli strain, the bacterial load increased rapidly, triggering an intense influx of leukocytes into mammary tissue and increased concentrations of IL-6, IL-22, TNF-α, and IL-10. Neutrophils were the first cells that migrated intensely to the mammary tissue, in line with an early production of CXCL2. Depletion of neutrophils induced an increased mammary bacterial load. There was a significant increase of IL-17–containing CD4+ αβ T lymphocyte numbers in infected glands. Depletion of IL-17A correlated with an increased bacterial colonization and IL-10 production. Intramammary infusion of IL-17A at the onset of infection was associated with markedly decreased bacterial numbers, decreased IL-10 production, and increased neutrophil recruitment. Depletion of CD25+ regulatory T cells correlated with a decreased production of IL-10 and a reduced bacterial load. These results indicate that IL-17A is an important effector of MG immunity to E. coli and suggest that an early increased local production of IL-17A would improve the outcome of infection. These findings point to a new lead to the development of vaccines against mastitis.

International audience; We studied the inflammatory and immune responses of bovine mammary epithelial cells (bMEC) infected by mastitis isolates of Staphylococeus aureus. Primary cultures of bMEC were co-incubated separately with three strains of S. aureus and one strain of Escherichia coli. Transcriptional levels and/or protein release of interleukin-8 (IL-8), growth related oncogene alpha (GRO-alpha), growth related ;oncogene beta (GRO-beta), tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), transforming growth factor beta 1 (TGF-beta 1) and interleukin-10 (IL-10) were measured at 3, 10 and 24 h post-infection (PI). The results indicated that at earlier hours of co-culture, bMEC infected with S. aureus or E. coli expressed more IL-1 beta, TNF-alpha, IL-8 and GRO-alpha mRNA than uninfected bMEC. Furthermore, infected bMEC released more TNF-alpha, IL-8, GRO-alpha and GRO-beta proteins than uninfected bMEC. However, differential transcription and release of some cytokines/chemokines from bMEC was observed according to the strain of S. aureus and bacteria Gram type. In conclusion, bMEC did not show an anti-inflammatory potential through IL-10 or TGF-beta 1 release. Nevertheless, bMEC were able to release neutrophil-mobilizing chemokines and pro-inflammatory cytokines upon bacterial stimulation, strongly suggesting that bMEC are active contributors to immune and inflammatory responses of mammary gland. In addition, the clinical characteristics and resolution of mastitis may be partly determined by the responses of bMEC according to S. aureus strains and bacteria Gram type. (c) 2007 Published by Elsevier Ltd.

Mastitis remains a major disease of cattle with a strong impact on the dairy industry. There is a growing interest in understanding how cell mediated immunity contributes to the defence of the mammary gland against invading mastitis causing bacteria. Cytokines belonging to the IL-17 family, and the cells that produce them, have been described as important modulators of the innate immunity, in particular that of epithelial cells. We report here that expression of IL-17A and IL-17F genes, encoding two members of the IL-17 family, are induced in udder tissues of cows experimentally infected with Escherichia coli. The impact of IL-17A on the innate response of bovine mammary epithelial cells was investigated using a newly isolated cell line, the PS cell line. We first showed that PS cells, similar to primary bovine mammary epithelial cells, were able to respond to agonists of TLR2 and to LPS, provided CD14 was added to the culture medium. We then showed that secretion of CXCL8 and transcription of innate immunity related-genes by PS cells were increased by IL-17A, in particular when these cells were stimulated with live E. coli bacteria. Together with data from the literature, these results support the hypothesis that IL-17A and IL-17 F could play an important role in mediating of host-pathogen interactions during mastitis. Electronic supplementary material The online version of this article (doi:10.1186/s13567-015-0201-4) contains supplementary material, which is available to authorized users.

Remerciements au LEGTA de Vendôme, Loir-et-Cher; Intramammary infusion of the antigen used to sensitize cows by the systemic route induces a local inflammation associated with neutrophil recruitment. We hypothesize that this form of delayed type hypersensitivity, which may occur naturally during infections or could be induced intentionally by vaccination, can impact the outcome of mammary gland infections. We immunized cows with ovalbumin to identify immunological correlates of antigen-specific mammary inflammation. Intraluminal injection of ovalbumin induced a mastitis characterized by a prompt tissue reaction (increase in teat wall thickness) and an intense influx of leukocytes into milk of 10 responder cows out of 14 immunized animals. The magnitude of the local inflammatory reaction, assessed through milk leukocytosis, correlated with antibody titers, skin thickness test, and production of IL-17A and IFN-γ in a whole-blood antigen stimulation assay (WBA). The production of these two cytokines significantly correlated with the magnitude of the milk leukocytosis following the ovalbumin intramammary challenge. The IL-17A and IFN-γ production in the WBA was dependent on the presence of CD4+ cells in blood samples. In vitro stimulation of peripheral blood lymphocytes with ovalbumin followed by stimulation with PMA/ionomycin allowed the identification by flow cytometry of CD4+ T cells producing either IL-17A, IFN-γ, or both cytokines. The results indicate that the antigen-specific WBA, and specifically IL-17A and IFN-γ production by circulating CD4+ cells, can be used as a predictor of mammary hypersensitivity to protein antigens. This prompts further studies aiming at determining how Th17 and/or Th1 lymphocytes modulate the immune response of the mammary gland to infection.

Chapitre 116; International audience; The mammary gland (MG) lacks a mucosa but is part of the mucosal immune system because of its role in passive mucosal immunity. The MG is not an inductive site for mucosal immunity. Rather, synthesis of immunoglobulin (Ig)A by plasma cells stimulated at distal inductive sites dominate in the milk of rodents, humans, and swine whereas IgG1 derived from serum predominates in ruminants. Despite the considerable biodiversity in the role of the MG, IgG passively transfers the maternal systemic immunological experience whereas IgA transfers the mucosal immunological experience. Although passive antibodies are protective, they and other lacteal constituents can be immunoregulatory. Immune protection of the MG largely depends on the innate immune system; the monocytes–macrophages group together with intraepithelial lymphocytes is dominant in the healthy gland. An increase in somatic cells (neutrophils) and various interleukins signal infection (mastitis) and a local immune response in the MG. The major role of the MG to mucosal immunity is the passive immunity supplied to the suckling neonate.

The C5a complement fragment is a potent inflammatory molecule but its contribution to the inflammatory response of the mammary gland remains uncertain. One of the unresolved questions is the possible interference of whole milk with C5a. In this study, the chemotactic activity ofpurifled bovine C5a was tested in the presence of whole or skimmed milk. Milk from healthy glands acted as a chemoattractant, which could mask any inhibitory effect on C5a activity. To circumvent milk activity, washed milk fat globules were incubated with optimal (1 nM) or suboptimal (0.1 nM) concentrations of C5a, and the eventual chemoattractant activity was measured. There was no reduction in C5a-induced migration through a polycarbonate filter or shape-change of neutrophils. The concentrations of C5a determined by ELISA in the fluid phase were not reduced after incubation with the fat globules. It can be concluded that bovine milk fat globules do not trap or degrade C5a. Although these results do not explain the inhibitory effect of whole milk in the C5a-induced recruitment of neutrophils in milk, they suggest that milk does not inhibit the second major activity of C5a (apart from chemotaxis), i.e. the stimulation of phagocytic and bactericidal activities of neutrophils.

International audience; The outer membrane protein (Omp) A is a major constituent of the outer membrane of Escherichia coli. This protein has been used in several vaccine development studies, but seldom with a view to vaccinating against mastitis. The objective of this study was to investigate the immunogenicity of E. coli OmpA and its vaccine potential for cows. Both the humoral and cellular immune responses were investigated. The gene for OmpA of the mastitis-causing strain P4 was cloned and expressed, and the recombinant protein (rEcOmpA) purified. Cows were immunized twice with rEcOmpA with adjuvant one month apart by the systemic route. Before immunization, few antibodies to rEcOmpA were detected, and there was little production of IL-17A in a whole blood stimulation assay (WBA) with rEcOmpA. Antibodies to rEcOmpA were induced by immunization. These antibodies were not able to react with E. coli P4, but reacted with a rough P4 mutant prepared by inactivating the rfb locus. This suggests that the complete LPS O-chain precluded the accessibility of antibodies to their target at the outer membrane. The cellular immune response appeared to be biased towards a Th17-type, as more IL-17A than IFN-γ was produced in the OmpA-specific WBA. There was a good correlation between antibody titers and the production of IL-17A in the WBA. The intramammary instillation of rEcOmpA elicited a slight local inflammatory response which was not related to the WBA. Overall, the interest of OmpA as vaccine immunogen was not established, although other experimental conditions (dose, adjuvant, route) need to be investigated to conclude definitively. The study pointed to several important issues such as the accessibility of OmpA to antibodies and the weakness of Th1-type response induced by OmpA.

International audience; Clinical mastitis caused by E. coli accounts for significant production losses and animal welfare concerns on dairy farms worldwide. The benefits of therapeutic intervention in mild to moderate cases are incompletely understood. We investigated the effect of intramammary treatment with cefapirin alone or in combination with prednisolone on gene expression profiles in experimentally-induced E. coli mastitis in six mid-lactating Holstein Friesian cows. Cows were challenged with E. coli in 3 quarters and received 4 doses of 300 mg cefapirin in one quarter and 4 doses of 300 mg cefapirin together with 20 mg prednisolone in another quarter. At 24 h (n = 3) or 48 h (n = 3) post-challenge, tissue samples from control and treated quarters were collected for microarray analysis. Gene expression analysis of challenged, un-treated quarters revealed an up-regulation of transcripts associated with immune response functions compared to un-challenged quarters. Both treatments resulted in down-regulation of these transcripts compared to challenged, un-treated quarters most prominently for genes representing Chemokine and TLR-signaling pathways. Gene expression of Lipopolysaccharide Binding Protein (LBP), CCL2 and CXCL2 were only significantly down-regulated in cefapirin-prednisolone-treated quarters compared to un-treated controls. Down-regulation of chemokines was further confirmed on the basis of protein levels in milk whey for CXCL1, CXCL2 and CXCL8 in both treatments with a greater decrease in cefapirin-prednisolone-treated quarters. The data reveal a significant effect of treatment on cell recruitment with a more pronounced effect in cefapirin-prednisolone treated quarters. Provided a rapid bacteriological clearance, combination therapy may prevent neutrophil-induced tissue damage and promote recovery of the gland.

We conducted the following study to determine if bispecific antibodies enhance the bactericidal activity of bovine neutrophils. Bispecific antibodies were synthesized by chemically crosslinking bovine neutrophil monoclonal antibodies to Staphylococcus aureus 305 capsule polysaccharide monoclonal antibodies. The efficiency of chemically coupling monoclonal antibody monomers was approximately 50% for each bispecific antibody produced. Monoclonal antibodies against neutrophils enhanced the respiratory burst activity of neutrophils by 2.3- to 2.5-fold. To determine the influence of bispecific antibodies on neutrophil function, S. aureus 305 was preincubated with various concentrations of bispecific antibodies and neutrophils were then added to the opsonized bacteria at different bacteria to neutrophil ratios. The bactericidal activity of neutrophils was expressed as a percentage reduction in colony-forming units in test cultures compared with the number of colony-forming units in control test cultures that did not contain bispecific antibodies or neutrophils. The addition of bispecific antibodies to test cultures increased the bactericidal activity of neutrophils. A reduction in colony-forming units as a function of increasing the S. aureus 305 to neutrophils ratio was observed in both the absence and presence of bispecific antibodies. However, a greater reduction was observed in the presence of bispecific antibodies. Increasing concentrations of bispecific antibodies enhanced the bactericidal activity of neutrophils at a constant S. aureus 305 to neutrophil ratio of 1:500. The results indicate that bispecific antibodies that recognize both S. aureus 305 capsular polysaccharide and neutrophil antigens potentiate the bactericidal activity of neutrophils.

Sequential changes in milk protein composition after experimental Escherichia coli mastitis. Milk protein composition was determined between 0 and 72 h after infection during experimentally induced E. coli mastitis of seven cows. In milk from infected quarters, contents of non protein nitrogen, total proteins and soluble proteins were significantly higher after 14 h and a max- imum was reached at 48 h after infection (+44 %, + 104 % and +574 % respectively). Casein content independently from time was reduced of 20 %. Proportions of 13- and œ-caseins were significantly lower at 24 h (-49 %) and between 48 and 72 h (--D2 %) respectively. Proportion of pH 4.6 insoluble pep- tides including y-caseins reached a maximum between 24 and 72 h (+580 %). These different kinet- ics can be explained by sequential origin ofproteolysis; plasmin activity could be maximal before max- imal activity of somatie cell proteases. Protein composition in milk from control quarters was also greatly modified. Regardless of time, soluble protein content increased (+68 %) and casein content decreased (-21 %). Significative proteolysis of l3-casein occurred at 72 h (-25 %) accompanied by increase in relative proportion of pH 4.6 insoluble peptides (+50 %); this was probably due to higher milk plasmin activity levels. Therefore, interdependance was notieed between infected and unin- fected quarters, probably through systemic response to E. coli infection. © InralElsevier, Paris.

It was reported previously that two capsular polysaccharides, types 5 and 8 (CP5 and CP8), account for 70 to 80% of Staphylococcus aureus strains isolated from human and animal sources. The capsular material has been shown to play a part in virulence and in resistance to phagocytosis. With a view to investigating the role that CP plays in pathogenicity or protection, relative measurement of cell-associated CP is desirable. Flow cytometry, which permits the analysis of individual bacteria, was used to that end. Thirty isolates expressing CP5, of human (n = 7) and animal (cow, n = 11; goat, n = 3; swine, n = 3; hen, n = 3; and rabbit, n = 3) origin, were cultivated on either brain heart infusion agar (BHI) or modified medium 110 (mod 110) agar. Staphylococci were incubated with a mouse anti-CP5 monoclonal antibody (an immunoglobulin M, which does not react with staphylococcal protein A) and then stained with a fluorescein-labeled anti-murine antibody. The bacteria were washed, sonicated, and analyzed by flow cytometry. Except for three isolates, the expression of cell-bound CP5 was higher when bacteria were cultivated on mod 110 than when they were cultivated on BHI. We found a wide intraisolate phenotypic heterogeneity in surface-exposed CP5 in many strains, which appeared as mixtures of stained and unstained bacteria. Four main patterns could be distinguished on the basis of the distribution of the fluorescence of individual bacteria within the strain population as a function of growth medium. Great variations in both percentages of stained bacteria and fluorescence intensity were recorded among strains regardless of their origin. Flow cytometry analysis provided information on both the relative amounts and the distribution patterns of the surface expression of CP. This information is potentially useful for the evaluation of the part played by the capsule in the interaction of bacteria with host cells or for the study of the activities of antibodies to this target antigen, such as opsonization or prevention of adherence.

International audience; Infectious mastitis cuts down milk production profitability and is a major animal welfare problem. Bacteria-induced inflammation in the mammary gland (MG) is driven by innate immunity, but adaptive immunity can modulate the innate response. Several studies have shown that it is possible to elicit inflammation in the MG by sensitization to an antigen subsequently infused into the lumen of the gland. The objective of our study was to characterize the inflammation triggered in the MG of cows sensitized to ovalbumin, by identifying the cytokines and chemokines likely to play a part in the reaction. Among immunized cows, responders mobilized locally high numbers of leukocytes. An overexpression of the genes encoding IL-17a, IL-17F, IL-21, IL-22 and INF-γ was found in milk cell RNA extracts in the early phase of the inflammatory response. At the protein level, IL-17A was detected in milk as soon as the first sampling time (8 h post-challenge), and both IL-17A and IFN-γ concentrations peaked at 12 to 24 h post-challenge. In mammary tissue from challenged quarters, overexpression of the genes encoding IL-17A, IL-17F, IL-21, IL-22, IL-26 and IFN-γ was observed. Neutrophil-attracting chemokines (CXCL3 and CXCL8) were found in milk, and overexpressed transcripts of chemokines attracting lymphocytes and other mononuclear leukocytes (CXCL10, CCL2, CCL5, CCL20) were detected in mammary tissue. Expression of IL-17A, as revealed by immunohistochemistry, was located in epithelial cells, in leukocytes in the connective tissue and in association with the epithelium, and in migrated alveolar leukocytes of challenged quarters. Altogether, these results show that antigen-specific inflammation in the MG was characterized by the production of IL-17 and IFN-γ. The orientation of the inflammatory response induced by the antigen-specific response has the potential to strongly impact the outcome of bacterial infections of the MG.