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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.

International audience; Interleukin-17 (IL-17) is a proinflammatory cytokine produced by activated memory T cells, and it appears to play an upstream role in T cell-triggered inflammation by stimulating stromal cells to secrete other cytokines. We hypothesize that IL-17 plays a role in the recruitment of neutrophils in the bovine mammary gland during infection or immune-mediated inflammation. The rapid amplification of cDNA ends (RACE) method was used to obtain a cDNA of bovine IL-17 (BoIL-17) containing a 462-bp open reading frame (ORF) encoding a protein of 153 amino acids (aa) with a molecular mass of 17.2 kDa, a 23-residue NH2-terminal signal peptide, a single potential N-linked glycosylation site, and 6 cysteine residues. BoIL-17 protein shared 73.5% identity with the human protein and 67% with the mouse and rat proteins. Sf9 insect cells were transfected with BoIL-17 cDNA, and supernatant was tested for biologic activity on a primary culture of bovine mammary epithelial cells (MECs). mRNA synthesis of IL-6, IL-8, and growth-related oncogene alpha (Gro alpha) was induced, suggesting a functional role for IL-17 in mammary immunity.

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; Decryption of diseases such as mastitis or breast cancer relies on our knowledge of the physiology of mammary epithelial cells. These cells produce milk, but they are also supposed to be sentinels for the detection of infections and the triggering of inflammation within the udder. Despite their pivotal role, none of the existing cell lines is sufficient to provide a steady and unfailing deciphering of the general physiology of mammary cells and their implication in immune responses against diseases. Here, we describe the PS cell line, especially its cellular purity and specificity, and its ability to respond to mastitis pathogens. Contrary to bovine primary cell cultures, the PS cell line is exclusively composed of luminal cytokeratin18-positive mammary epithelial cells. It expresses α and β-caseins, and it is able to secrete cytokines, chemokines (CCL20, CXCL8) and anti-microbial peptides (SAA3, TAP) in response to bacterial agonists in the absence of serum, unlike other mammary epithelial cell lines. This immune capacity is allowed by expression of functional receptors implicated in the recognition of components synthetized by mastitis pathogens, in particular lipopeptides (recognized by toll-like receptors (TLR) 1, 2 and 6). Other Microbe-Associated Molecular Patterns (MAMPs) such as peptidoglycan subunits (NOD 1 or NOD2), flagellin (TLR5), and LPS (TLR4-CD14-MD2 complex) induce weaker responses. This may result from a low expression of TLR5 and CD14, in keeping with some in vivo data but at variance with some data obtained with primary cells. The PS cell line will be useful to specify the part played by mammary epithelial cells in the innate defense against mastitis, and to investigate their interplay with the other cell populations constituting the udder during mastitis.

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.

National audience; En France et en Europe, les mammites constituent la première source de pertes financières des cheptels bovins laitiers et allaitants. Il s’agit d’une inflammation de la glande mammaire, essentiellement d’origine bactérienne. Les infections induites par Escherichia coli (E. coli) s’accompagnent de signes cliniques de gravité variable, et sont en général brèves, du fait d’une clairance rapide du pathogène. Néanmoins dans certains cas, la bactérie entraîne une réaction systémique chez la vache, pouvant altérer la fonctionnalité de la mamelle voire induire la mort de l’animal. Ainsi la présente étude vise-t-elle à caractériser et à comprendre comment des souches colibacillaires peuvent induire des réponses de la glande mammaire diverses. Les résultats portant sur quatre souches d’E. coli isolées de cas de mammites tendent à montrer que les réponses inflammatoires déclenchées par ces souches sont très différentes en termes de recrutement cellulaire et de production de cytokines pro-inflammatoires, de chimiokines et de peptides antimicrobiens. Des caractéristiques intrinsèques telles que la résistance au sérum bovin, une capacité accrue de multiplication dans le lait et un faible pouvoir inducteur de réponse immunitaire des cellules épithéliales mammaires pourraient expliquer comment des souches d’E. coli sont capables d’induire des mammites aiguës et suraiguës. Au contraire, des souches hautement sensibles au sérum, se multipliant faiblement dans le lait et déclenchant des réponses inflammatoires fortes seraient rapidement éliminées de la glande mammaire, sans induction de signe clinique. Cette étude souligne ainsi l’importance de considérer les mammites en fonction des signes cliniques observés, et en regard des souches bactériennes impliquées.

The mammary gland is able to detect and react to bacterial intrusion through innate immunity mechanisms, but mammary inflammation can also result from antigen-specific adaptive immunity. We postulated that innate and adaptive immune responses could synergize to trigger inflammation in the mammary gland. To test this hypothesis, we immunized cows with the model antigen ovalbumin and challenged the sensitized animals with either Escherichia coli lipopolysaccharide (LPS) as innate immunity agonist, ovalbumin as adaptive immunity agonist, or both agonists in three different udder quarters of lactating cows. There was a significant amplification of the initial milk leukocytosis in the quarters challenged with the two agonists compared to leukocytosis in quarters challenged with LPS or ovalbumin alone. This synergistic response occurred only with the cows that developed the ovalbumin-specific inflammatory response, and there were significant correlations between milk leukocytosis and production of IL-17A and IFN-γ in a whole-blood ovalbumin stimulation assay. The antigen-specific response induced substantial concentrations of IL-17A and IFN-γ in milk contrary to the response to LPS. Such a synergy at the onset of the reaction of the mammary gland suggests that induction of antigen-specific immune response with bacterial antigens could improve the initial immune response to infection, hence reducing the bacterial load and contributing to protection.

National audience

Escherichia coli is a frequent cause of clinical mastitis in dairy cows. It has been shown that a prompt response of the mammary gland after E. coli entry into the lumen of the gland is required to control the infection, which means that the early detection of bacteria is of prime importance. Yet, apart from lipopolysaccharide (LPS), little is known of the bacterial components which are detected by the mammary innate immune system. We investigated the repertoire of potential bacterial agonists sensed by the udder and bovine mammary epithelial cells (bMEC) during E. coli mastitis by using purified or synthetic molecular surrogates of bacterial agonists of identified pattern-recognition receptors (PRRs). The production of CXCL8 and the influx of leucocytes in milk were the readouts of reactivity of stimulated cultured bMEC and challenged udders, respectively. Quantitative PCR revealed that bMEC in culture expressed the nucleotide oligomerization domain receptors NOD1 and NOD2, along with the Toll-like receptors TLR1, TLR2, TLR4, and TLR6, but hardly TLR5. In line with expression data, bMEC proved to react to the cognate agonists C12-iE-DAP (NOD1), Pam3CSK4 (TLR1/2), Pam2CSK4 (TLR2/6), pure LPS (TLR4), but not to flagellin (TLR5). As the udder reactivity to NOD1 and TLR5 agonists has never been reported, we tested whether the mammary gland reacted to intramammary infusion of C12-iE-DAP or flagellin. The udder reacted to C12-iE-DAP, but not to flagellin, in line with the reactivity of bMEC. These results extend our knowledge of the reactivity of the bovine mammary gland to bacterial agonists of the innate immune system, and suggest that E. coli can be recognized by several PRRs including NOD1, but unexpectedly not by TLR5. The way the mammary gland senses E. coli is likely to shape the innate immune response and finally the outcome of E. coli mastitis.

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

Staphylococcus aureus colonizes and infects humans as well as animals. In the present study, 17 S. aureus strains isolated from cows suffering from mastitis were characterized. The well-established multilocus sequence typing (MLST) technique and a diagnostic microarray covering 185 S. aureus virulence and resistance genes were used for genetic and epidemiological analyses. Virulence gene expression studies were performed by analyzing the extracellular protein pattern of each isolate on 2-D gels. By this way, a pronounced heterogeneity of the extracellular proteome between the bovine isolates has been observed which was attributed to genome plasticity and variation of gene expression. Merely 12 proteins were expressed in at least 80% of the isolates, i.e. Atl, Aur, GlpQ, Hla, LtaS, Nuc, PdhB, SAB0846, SAB2176, SAB0566, SspA, and SspB forming the core exoproteome. Fifteen extracellular proteins were highly variably expressed and only present in less than 20% of the isolates. This includes the serine proteases SplB, C, and F, and the superantigens SEC-bov, SEL and TSST-1. Compared to human isolates we identified at least six proteins with significantly different expression frequencies. While SAB0846 was expressed more frequently in bovine isolates, LytM, EbpS, Spa, Geh, and LukL1 were seen less frequently in these isolates.

International audience; An early recruitment of neutrophils in mammary tissue and milk is considered an important component of the defense of the mammary gland against Staphylococcus aureus. We investigated whether the leukotoxin LukM/F', which is produced by a proportion of mastitis-causing strains of S. aureus, would be able to trigger inflammation in the udder. Infusion of purified LukM/F' toxin in lactating mammary glands did not cause neutrophil influx in milk, showing that the toxin was not able to cause mastitis on its own. Purified LukM/F' did not kill or stimulate mammary epithelial cells in culture. As expected, LukM bound to mammary macrophages and the complete LukM/F' toxin killed these cells, but subcytotoxic LukM/F' concentrations did not induce secretion of IL-8, TNF-alpha, IL-1 beta or IL-6 by macrophages. On the contrary, the production of these pro-inflammatory mediators by adhesion-stimulated macrophages was reduced. Overall, these results indicate that purified leukotoxin LukM/F' is not likely to contribute to the initiation of the inflammatory response and could even play an anti-inflammatory role in the mammary gland by inactivating macrophages

In the rapidly changing context of research on animal health, INRA launched a collective discussion on the challenges facing the field, its distinguishing features, and synergies with biomedical research. As has been declared forcibly by the heads of WHO, FAO and OIE, the challenges facing animal health, beyond diseases transmissible to humans, are critically important and involve food security, agriculture economics, and the ensemble of economic activities associated with agriculture. There are in addition issues related to public health (zoonoses, xenobiotics, antimicrobial resistance), the environment, and animal welfare. Animal health research is distinguished by particular methodologies and scientific questions that stem from the specific biological features of domestic species and from animal husbandry practices. It generally does not explore the same scientific questions as research on human biology, even when the same pathogens are being studied, and the discipline is rooted in a very specific agricultural and economic context. Generic and methodological synergies nevertheless exist with biomedical research, particularly with regard to tools and biological models. Certain domestic species furthermore present more functional similarities with humans than laboratory rodents. The singularity of animal health research in relation to biomedical research should be taken into account in the organization, evaluation, and funding of the field through a policy that clearly recognizes the specific issues at stake. At the same time, the One Health approach should facilitate closer collaboration between biomedical and animal health research at the level of research teams and programmes.

International audience; Interleukin-17A (IL-17A) and IL-17F have been shown to mediate a crucial crosstalk between the immune system and various epithelial tissues, stimulating various defensive mechanisms to bacterial infections. A number of studies have characterized the response to IL-17A and IL-17F of epithelial cells from airways, intestine, and skin, but not from the mammary gland. To evaluate the potential contribution of IL-17 to the immune defense of the mammary gland, we analyzed the effects of recombinant bovine IL-17A and IL-17F on primary bovine mammary epithelial cells (MEC) by quantitative PCR and ELISA. We found expression (mRNA) of the two components of the IL-17 receptor complex, IL-17RA and IL-17RC, in mammary tissue and MEC in vitro. The expression of a number of genes encoding cytokines, chemokines and proteins endowed with antibacterial activities was increased by IL-17A, and to a lesser extent by IL-17F, but the magnitude of responses was modest. As expected, responses were augmented by the combination of IL-17A or IL-17F with TNF-a. Interestingly, responses of a few of the tested genes, such as IL8, CCL20, iNOS, and CfB, were augmented by the combination of IL-17A with staphylococcal lipoteichoic acid or muramyl dipeptide, bacterial agonists of the innate immune system. This can be interpreted as indicating that IL-17A and IL-17F are tailored to exert their full potential in a septic environment. MEC responses were characterized by the expression of chemokines targeting not only neutrophils (CXCL3 and CXCL8) but also mononuclear leucocytes (CCL2, CCL20). Production of IL-6 was low and the inflammatory cytokines TNF-a and IL-1b were expressed (mRNA) but proteins were not secreted. Altogether, our results suggest that IL-17A and IL-17F have a potential to modulate the mammary gland immune response to mastitis-causing pathogens.

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.

Mammary epithelial cells (MEC) participate in the first line of defense of the mammary gland to invading pathogens. In vitro culture of MEC is widely used as a model to study the capacity of these cells to sense and respond to mastitis-causing bacteria. Analysis of gene expression by quantitative PCR (qPCR) following exposure to bacteria or bacterial constituents is a powerful tool to assess responses of MEC to pathogens. Although internal standards such as reference genes are required for qPCR to yield valid data, the validation of proper genes to quantify mRNA transcripts in MEC exposed to pro-inflammatory stimuli has never been reported. In this study, 10 commonly used reference genes belonging to different functional classes (ACTB, ATP5B, EIF2B2, GAPDH, PPIA, SDHA, SUZ12, UXT, YWHAZ, and 18s rRNA) were analyzed by qPCR to determine the most stable in bovine MEC unstimulated and stimulated with mastitis pathogens (Staphylococcus aureus or Escherichia coli), microbial agonists of the innate immune system (lipoteichoic acid and muramyl dipeptide, or lipopolysaccharide), or proinflammatory cytokines (IL-17A and tumor necrosis factor-α). An M value was used as a measure of gene stability as determined using the geNorm application. This study demonstrated that the expression of the 10 reference genes was stable under the different experimental conditions. These data will be useful for bovine mastitis research in selecting reference genes and validating reverse transcription-qPCR data.

It is well known that signaling in neutrophils through both the complement component 5a (C5a) and C5a receptor (C5aR) and the toll-like receptor 4 (TLR4) pathways plays an essential role in innate defense. Neutrophil dysfunction, as seen during sepsis in severe mastitis during the periparturient period, is correlated with elevated concentrations of anaphylatoxin C5a. The aim of the current study was to elucidate the effect of C5a on TLR4 signaling in bovine neutrophils. Neutrophils were incubated with a high (but physiological) dose of purified C5a, and mRNA was extracted from neutrophils at different time points postincubation (PI). The incubation with C5a resulted in a biphasic C5aR expression profile, a phenomenon that might be explained by internalization (at 10 min PI) with subsequent reconstitution (starting at 40 min PI) of this receptor. The expression of TLR4, as well as its coreceptor, CD14, showed a similar biphasic change as observed with C5aR. In addition, changes in the mRNA expression levels of several genes belonging to the TLR4 pathway, such as TICAM-1, IKKα, and MAP3K7 were noted. The maximal expression of TLR4, CD14, and C5aR mRNA at 80 min PI was accompanied by a peak in IL8 mRNA, indicating that C5a is able to induce IL-8 production in neutrophils in vitro without the need of a costimulatory factor such as lipopolysaccharide. Moreover, a relatively constant expression of RELA was accompanied by increased expression of ATF3, an endogenous inhibitor of nuclear factor-κB mediated transcription, implying that C5a regulates TLR4 signaling and IL-8 synthesis independently. A significant time-dependent correlation was found between C5aR and TLR4, with the majority of the selected TLR4-dependent genes showing a significant correlation with C5aR at 80 min PI, when C5aR and TLR4 mRNA expression reached its maximum, suggesting crosstalk between both receptors. Taken together, this study showed that C5a is able to (1) alter the expression of genes belonging to the TLR4 pathway and (2) induce IL8 gene expression in bovine neutrophils. In addition, indications for cross-talk between complement activation and TLR4 signaling were found in the present study.

International audience; The range of leucocytes susceptible to the leucotoxin LukM/F', a two-component pore-forming toxin of Staphylococcus aureus causing mastitis in ruminants, had not been defined. We used fluorescent-labeled LukM to investigate the binding of this toxin to bovine cells and to identify its cellular targets among bovine, human and murine leucocytes. LukM bound to bovine blood neutrophils from all the individuals tested with similar affinity, with an apparent dissociation constant (Kd) of 1.81 +/- 0.14 nM and 13 3100 +/- 506 binding sites. The amount of LukM bound to bovine neutrophils did not depend on the presence of the complementary component LukF, suggesting that the binding of LukM to its ligand does not depend on the formation of pore-forming oligomers, and that the number of bound LukM molecules corresponds to the number of available cell membrane ligands. Other staphylococcal class S components of bipartite leucotoxins (HIgA, HIgC, LukE, LukS-PV) were inefficient competitors of LukM for the binding to bovine neutrophils, indicating that LukM has a distinct ligand on target cells. Bovine blood neutrophils bound slightly more LukM than did milk neutrophils, and much more than did ovine and caprine blood neutrophils. Bovine monocytes and milk macrophages readily bound LukM, whereas blood lymphocytes did not. Human neutrophils bound little LukM and were resistant to LukM/F' at the highest tested concentration (40 nM). Murine neutrophils bound LukM and were susceptible to the toxicity of LukM/F', exhibiting flattening and nucleus alteration beginning at 0.3 nM concentration. Among murine peritoneal exudate cells, T lymphocytes (CD3+) and monocytes/macrophages (F4/80+) bound LukM, whereas binding to B lymphocytes (CD19+) was not detected. These results indicate that cells of the myeloid lineage are the main targets of LukM/F' in dairy ruminants, and that resident or inflammatory migrated phagocytes are susceptible to this toxin. (C) 2010 Elsevier Ltd. All rights reserved.

Dans le contexte très évolutif de la recherche en santé animale, une réflexion sur ses enjeux, ses spécificités et ses synergies avec la recherche biomédicale, a été conduite à l’initiative de l’INRA. Affirmés au premier chef par l’OMS, la FAO et l’OIE, les enjeux en santé animale, hors des maladies transmissibles à l’Homme, sont énormes et touchent à la sécurité alimentaire, l’économie de l’agriculture et l’ensemble des activités économiques qui en découlent. S’y ajoutent les enjeux de santé publique (zoonoses, xénobiotiques, antibiorésistance), environnementaux et de bien-être animal. La recherche en santé animale présente des spécificités d’ordre méthodologique et scientifique, liées notamment aux particularités biologiques des espèces domestiques et aux pratiques d’élevage. Elle n’a pas les mêmes questionnements scientifiques qu’en biologie humaine même lorsqu’elle traite des mêmes agents pathogènes et, connectée aux autres sciences animales (génétique, physiologie, zootechnie), elle s’enracine dans une réalité agricole et économique très spécifique. Des synergies génériques et méthodologiques existent néanmoins avec la recherche biomédicale, en particulier autour des outils et des modèles biologiques. Certaines espèces domestiques (tel le porc) présentent en outre des similitudes fonctionnelles avec l’Homme, plus que le rongeur de laboratoire. Ainsi la singularité de la recherche en santé animale par rapport à la recherche en biologie humaine devrait être prise en compte dans son organisation, son évaluation et son financement, via une politique de reconnaissance des enjeux spécifiques. Simultanément, l’approche one health devrait faciliter une collaboration approfondie entre recherche en biologie humaine et recherche en santé animale, à l’échelle des équipes ou des programmes., In the moving context of research on animal health, a collective discussion has been carried out on its issues, special features and synergism with biomedical research. Strongly enhanced by WHO, FAO and OIE, the issues of animal health are important and deal with food security, agriculture economics and all economical activities that follow from it. Others are related to public health (zoonoses, xenobiotics, antimicrobial resistance), environment and animal welfare. Research on animal health has got special features concerning methodology and scientific questions, among others the particular biological nature of domestic species and breeding practices. Scientific questions are not similar to those of biomedical research, even if dealing with the same pathogens; connected to the other animal sciences (genetics, physiology, animal breeding), research on animal health takes root in a very specific agricultural and economical reality. Nevertheless, generic and methodological synergies do exist with biomedical research, particularly concerning biological tools and models. Some domestic species like pig have functional similarities with Humans, better than with mouse or rat. Thus, the peculiarity of research on animal health compared to biomedical research should be considered in its organisation, evaluation and funding, through a political recognition of specific issues. Simultaneously, the one health approach should facilitate a thorough collaboration between biomedical research and research on animal health, at the levels of research teams and research programmes.

Concentrations of the chemoattractants CXCL1, CXCL2, CXCL3, CXCL8, and C5a in milk were reduced by the preparation of milk whey by high-speed centrifugation or with rennet. About half of the chemoattractants (35 to 65%) were associated with the casein micelle sediment, except when whey was prepared by acidification. Consequently, quantification of chemoattractants should be carried out preferentially with skimmed milk samples or, whenever whey is needed, with acidic whey samples. The interference of milk or milk whey with the enzyme-linked immunosorbent assays (ELISAs) used to quantify the chemoattractants was moderate, as long as tetramethylbenzidine (TMB), not ABTS [2,2′-azino-bis-(3-ethylbenzthiazoline-sulfonate)], was used as the substrate of peroxidase. These considerations will help to assess more precisely a component of the immune response of the mammary gland to infection.

Staphylococcus aureus , a major pathogen for the mammary gland of dairy ruminants, elicits the recruitment of neutrophils into milk during mastitis, but the mechanisms are incompletely understood. We investigated the response of the bovine mammary gland to muramyl dipeptide (MDP), an elementary constituent of the bacterial peptidoglycan, alone or in combination with lipoteichoic acid (LTA), another staphylococcal microbial-associated molecular pattern (MAMP). MDP induced a prompt and marked influx of neutrophils in milk, and its combination with LTA elicited a more intense and prolonged influx than the responses to either stimulus alone. The concentrations of several chemoattractants for neutrophils (CXCL1, CXCL2, CXCL3, CXCL8, and C5a) increased in milk after challenge, and the highest increases followed challenge with the combination of MDP and LTA. MDP and LTA were also synergistic in inducing in vitro chemokine production by bovine mammary epithelial cells (bMEpC). Nucleotide-binding oligomerization domain 2 (NOD2), a major sensor of MDP, was expressed (mRNA) in bovine mammary tissue and by bMEpC in culture. The production of interleukin-8 (IL-8) following the stimulation of bMEpC by LTA and MDP was dependent on the activation of NF-κB. LTA-induced IL-8 production did not depend on platelet-activating factor receptor (PAFR), as the PAFR antagonist WEB2086 was without effect. In contrast, bMEpC and mammary tissue are known to express Toll-like receptor 2 (TLR2) and to respond to TLR2 agonists. Although the levels of expression of the inflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-1β were increased by LTA and MDP at the mRNA level, no protein could be detected in the bMEpC culture supernatant. The level of induction of IL-6 was low at both the mRNA and protein levels. These results indicate that MDP and LTA exert synergistic effects to induce neutrophilic inflammation in the mammary gland. These results also show that bMEpC could contribute to the inflammatory response by recognizing LTA and MDP and secreting chemokines but not proinflammatory cytokines. Overall, this study indicates that the TLR2 and NOD2 pathways could cooperate to trigger an innate immune response to S. aureus mastitis.

Bovine lactoferrin is an iron-binding protein present in mammary gland secretions. The exposure of Streptococcus agalactiae to bovine lactoferrin resulted in the binding of this protein to all the 12 strains of bovine origin tested, and also, although to a lesser degree, to the five tested strains of human origin. The interaction of lactoferrin with one high-binding bovine strain (24/60, the prototype NT/X strain) was studied. Binding was time-dependent, dose-dependent, and saturable. The binding of lactoferrin was slightly affected by cultivation conditions, and appeared to be heat-stable. The binding of biotinylated lactoferrin was inhibited by unlabelled lactoferrin but not by bovine serum albumin.

The protein X of Steptococcus agalactiae is a surface antigen included in the typing scheme of group B streptococci (GBS). We have developed a monoclonal antibody to the protein X and used it to purify this antigen by affinity chromatography. Electrophoresis in polyacrylamide, and immunoblotting using the monoclonal antibody or a rabbit antiserum raised with the affinity purified protein X, revealed a major band in the region of 200 kDa and a smaller one at 100 kDa. The isolated protein X will make possible investigations of its potential role in virulence and protection.

Recruitment of phagocytes to inflammatory sites involves the coordinated action of several chemoattractants, including the anaphylatoxin C5a. While the C5a receptor (C5aR) has been well characterized in humans and rodents, little is known about the bovine C5aR. Here, we report cloning of bovine C5R1, the gene encoding bovine C5aR. We also analyzed genomic sequence upstream of the C5R1 translation start site. Although the bovine C5aR amino acid sequence was well conserved among species, significant differences in conserved features were found, including major differences in the N terminus, intracellular loop 3, and transmembrane domain VII. Analysis of C5aR expression by flow cytometry and confocal microscopy demonstrated high levels of C5aR on all bovine neutrophils and a subset of bovine monocytes. C5aR was not expressed on resting or activated bovine lymphocytes, although C5aR message was present in these cells. C5aR was also expressed on a small subset of bovine mammary epithelial cells. Pharmacological analysis of bovine C5aR-mediated responses showed that bovine C5a and C5adesArg both induced dose-dependent calcium fluxes and chemotaxis in bovine neutrophils, with similar efficacy for both agonists. Treatment of bovine neutrophils with C5a or C5adesArg resulted in homologous desensitization of bovine C5aR and cross-desensitization to interleukin 8 (IL-8) and platelet-activating factor (PAF); whereas, treatment with IL-8 or PAF did not cross-desensitize the cells to C5a or C5adesArg. Overall, these studies provide important information regarding distinct structural and functional features that may contribute to the unique pharmacological properties of bovine C5aR.

The response of the bovine mammary gland to lipoteichoic acid (LTA), which is a major pathogen-associated molecular pattern of Gram-positive bacteria, was investigated by infusing purified Staphylococcus aureus LTA in the lumen of the gland. LTA was able to induce clinical mastitis at the dose of 100g/quarter, and a subclinical inflammatory response at 10g/quarter. The induced inflammation was characterized by a prompt and massive influx of neutrophils in milk. LTA proved to induce strongly the secretion of the chemokines CXCL1, CXCL2, CXCL3 and CXCL8, which target mainly neutrophils. The complement-derived chemoattractant C5a was generated in milk only with the highest dose of LTA (100g). The pro-inflammatory cytokine IL-1 was induced in milk, but there was very little if any TNF- and no IFN-. The re-assessment of CXCL8 concentrations in milk whey of quarters previously challenged with S. aureus, by using an ELISA designed for bovine CXCL8, showed that this chemokine was induced in milk, contradicting previous reports. Overall, S. aureusLTA elicited mammary inflammatory responses that shared several attributes with S. aureus mastitis. Purified LTA looks promising as a convenient tool to investigate the inflammatory and immune responses of the mammary gland to S. aureus. Staphylococcus aureus / mastitis / cattle / lipoteichoic acid / chemokine

In the presence of milk, bovine polymorphonuclear cells (PMN) assumed a polarized shape, a feature of motile cells, and their adherence to plastic was augmented. Milk enhanced the phagocytosis of glass- or plastic-adherent Streptococcus agalactiae. In contrast, PMN were not stimulated by colostrum.

A total of 193 Staphylococcus aureus isolates from bovine, caprine, and ovine mastitis producing type 5 or 8 capsular polysaccharides were investigated for colony morphology in serum-soft agar and agglutinability by an anti-teichoic acid serum, after cultivation in modified staphylococcus medium no. 110. Also, 40 of these strains were cultivated in brain heart infusion and submitted to clumping factor and protein A detection tests. Considering capsular serotyping as a reference method, diffuse growth in serum-soft agar and inagglutinability by anti-teichoic acid serum identified, respectively, 57.5 and 45% of encapsulated strains cultivated in brain heart infusion and 85.5 and 77.2% of those cultivated in modified staphylococcus medium 110. Consequently, these indirect techniques underestimated encapsulation and were greatly influenced by culture conditions. Whatever the medium used, diffuse colony morphology in serum-soft agar was generally characterized by a masking of teichoic acid and protein A. By contrast, these surface antigens were detected in association with compact morphology; the presence of a thin or discontinuous capsular material could explain this result. Moreover, the masking of teichoic acid and the removal of capsular polysaccharide by washing in saline suggest that type 8 capsular polysaccharide is more abundant and labile than type 5.

Quarter foremilk samples were taken at 2-3 weekly intervals for several years in an experimental herd comprising about 45 cows. The samples were submitted to bacteriological analysis and somatic cell counting. The most prevalent quarter infections from 1982 to 1988 were by coagulase-negative staphylococci (15-20% of all the quarters sampled). Most of these (75.6%) persisted until drying-off. Dry cow therapy eliminated 86.5% of these infections. Comparison of udder quarters within cows, involving 775 samples from pairs of non-infected quarters and quarters infected by coagulase-negative staphylococci, yielded geometric means of somatic cell counts of 210,000 and 420,000 cells/ml, respectively. The correlation (r = 0.87) between the herd bulk milk somatic cell count (SCC) and its estimation from the quarter milk somatic cell count performed on the same day allowed us to evaluate the contribution of the different categories of quarters, according to their infection status, to the herd bulk milk SCC. Quarters infected by a major pathogen (8.5% of samples) gave rise to 46.6% of the total number of cells, while quarters infected by coagulase-negative staphylococci (17.8% of samples) gave rise to 18.1%. Although coagulase-negative staphylococci represented only a secondary source of somatic cells as compared to major pathogens, they were not a negligible source considering the threshold of 300,000 somatic cells advocated for herd milk of good quality.

We sought an explanation for epidemiological changes in Streptococcus agalactiae infections by investigating the link between ecological niches of the bacterium by determining the prevalence of 11 mobile genetic elements. The prevalence of nine of these elements differed significantly according to the human or bovine origin of the isolate. Correlating this distribution with the phylogeny obtained by multilocus sequence analysis, we observed that human isolates harboring GBSi1, a clear marker of the bovine niche, clustered in clonal complex 17. Our results are thus consistent with the emergence of this virulent human clone from a bovine ancestor.

Leucotoxins are regarded as virulence factors of Staphylococcus aureus, but data sup- porting their importance in infection are scarce. Experimental infections of the mammary glands of six goats with a leucotoxin-producing strain were used to investigate in vivo production of leu- cotoxin. Leucotoxin M/F' was monitored in milk as well as antibodies to LukM/F' in serum and milk. Leucotoxin antigen was detected by ELISA in milk samples of two goats. The appearance of neutrophils in these samples showed similarity with the altered appearance of neutrophils incubated with purified LukM/F' leucotoxin. Leucotoxic activity was found in the milk samples of these two goats, in particular those of the only goat which developed gangrenous (necrotic) mastitis, and this activity was inhibited by antibodies to LukM/F'. Increases in specific antibody titers occurred in the serum and milk of infected goats, and neutralizing titers increased in serum. The results indicate that the lukM and lukF' genes were functional in vivo, and actively transcribed and translated in the course of mammary infection. These results suggest that LukM/F' can interfere with neutrophil defense activities during infection, and they prompt further studies to investigate the contribution of leucotoxins to staphylococcal mastitis. Staphylococcus aureus / leucotoxin / mastitis / ruminants

International audience; Understanding the immune defenses of the mammary gland is instrumental in devising and developing measures to control mastitis, the major illness of dairy ruminants. Innate immunity is an extremely broad field for investigation, and despite decades of research, our present knowledge of the innate defenses of the udder is incomplete. Yet, information is being gained on the recognition of pathogens by the mammary gland, and on several locally inducible defenses. The contribution of mammary epithelial cells to local defenses and to the mobilization of leucocytes is under growing scrutiny. Interactions of mastitis-causing bacteria such as Escherichia coli or Staphylococcus aureus and the mammary gland represents a suitable model for studies on innate immunity at an epithelium frontier. Powerful new research tools are radically modifying the prospects for the understanding of the interplay between the mammary gland innate defenses and mastitis-causing bacteria: genetic dissection of the immune response, microarray gene technology, transcriptomic methodologies and gene silencing by RNA interference will make possible the discovery of several of the key defense mechanisms which govern the susceptibility/resistance to mastitis at the molecular and genetic levels. It should then be possible to enhance the resistance of dairy ruminants to mastitis through immunomodulation and genetic improvement.

International audience; Staphylococcus aureus is a ubiquitous pathogen causing infections in humans and domestic animals. It is often associated with bovine mastitis. Among secreted virulence factors, the leukotoxins constitute a family of toxins composed of two distinct subunits (class S and F proteins) which induce first Ca2+ influx and subsequent pore formation that allows ethidium entry. As mastitis-causing isolates harbor the genes of at least two, and often three leukotoxins, we compared the biological activities of the purified leukotoxins whose genes are found in mastitis-causing isolates on bovine polymorphonuclear neutrophils (PMN): spreading on a solid support, calcium influx and ethidium entry. In the spreading assay, the homologous pair LukM/LukF′-PV was the most active leukotoxin. Within each class, either S or F, subunits were interchangeable and generated leukotoxins with different specific activity. LukM was also very active when associated with heterologous F subunits. A similar ranking of homologous pairs was also found in the ethidium entry assay: LukM/LukF′-PV > HlgA/HlgB > HlgC/HlgB > LukE/LukD = LukEv/LukDv. In the Ca2+ flux assay, LukM/F′-PV was the most active pair, but γ-hemolysin (Hlg) was also very efficient. LukEv/Dv was more active (twofold) than LukE/D in the spreading assay, but the two variants showed similar activities in the other two assays. Supposing that spreading and ethidium entry (pore formation) reflect toxic activities on bovine PMN, and Ca2+ influx cell activation, LukM/F′-PV was by far the most cytotoxic leukotoxin, but it was closely followed by γ-hemolysin for PMN activation. These results suggest that LukM/F′-PV may constitute a particular virulence attribute of mastitis-causing S. aureus strains.

Transgenic cows expressing an antibacterial endopeptidase in their mammary glands show enhanced resistance to mastitis.

Acute Escherichia coli mastitis is one of the major sources of economic loss in the dairy industry due to reduced milk production, treatment costs, discarded milk, and occasional fatal disease. Nonsteroidal anti-inflammatory drugs ( NSAIDs) are frequently used as adjunctive therapy to antibiotics. The objective of the current study was to evaluate the effect of carprofen treatment following infusion of Escherichia coli into the mammary glands of primiparous cows during the periparturient period. Severity of mastitis was scored based on the average milk production in the uninfected quarters on d+2 postinoculation and a clinical severity score. Carprofen was administered intravenously at 9 h postchallenge, when clinical signs of mastitis appeared. In previous work, efficacy of NSAIDs was mainly evaluated using clinical symptoms. In the present study, the effect of carprofen on innate immune response was also assessed by quantification of inflammatory mediators. All primiparous cows reacted as moderate responders throughout the experimental period. Primiparous cows were intramammarily inoculated with 1x10(4) cfu of E. coli P4:O32 in 2 left quarters. Analysis of blood and milk parameters, including IL-8, complement component C5a, lipopolysaccharide-binding protein (LBP), soluble CD14, prostaglandin E-2, and thromboxane B-2 was performed from d0 to d+6 relative to intramammary inoculation. Rectal temperature in carprofen-treated animals was lower than in control animals at 3 and 6 h posttreatment. Treatment also restored the decreased reticulorumen motility that occurs during E. coli mastitis to preinfection levels faster than in control animals. Carprofen treatment resulted in an earlier normalization of the clinical severity score. Eicosanoid (prostaglandin E-2 and thromboxane B-2) production in milk tended to be inhibited by carprofen. No significant differences in the kinetic patterns of somatic cell count, IL-8, complement component C5a, LBP, and soluble CD14 were observed. In conclusion, carprofen treatment improved general clinical condition by effective antipyrexia and restoration of reticulorumen motility but did not significantly inhibit eicosanoid production. Carprofen treatment did not result in a significant decrease of chemotactic inflammatory mediators, IL-8 and C5a, and early innate immune molecules, sCD14 and LBP. Therefore, major modulatory effects from NSAID administration were not observed in this mastitis model, although a larger study might confirm some apparent trends obtained in the present results.

Staphylococcus aureusandEscherichia coliare among the most prevalent species of gram-positive and gram-negative bacteria, respectively, that induce clinical mastitis. The innate immune system comprises the immediate host defense mechanisms to protect against infection and contributes to the initial detection of and proinflammatory response to infectious pathogens. The objective of the present study was to characterize the different innate immune responses to experimental intramammary infection withE. coliandS. aureusduring clinical mastitis. The cytokine response and changes in the levels of soluble CD14 (sCD14) and lipopolysaccharide-binding protein (LBP), two proteins that contribute to host recognition of bacterial cell wall products, were studied. Intramammary infection with eitherE. coliorS. aureuselicited systemic changes, including decreased milk output, a febrile response, and induction of the acute-phase synthesis of LBP. Infection with either bacterium resulted in increased levels of interleukin 1β (IL-1β), gamma interferon, IL-12, sCD14, and LBP in milk. High levels of the complement cleavage product C5a and the anti-inflammatory cytokine IL-10 were detected at several time points followingE. coliinfection, whereasS. aureusinfection elicited a slight but detectable increase in these mediators at a single time point. Increases in IL-8 and tumor necrosis factor alpha were observed only in quarters infected withE. coli. Together, these data demonstrate the variability of the host innate immune response toE. coliandS. aureusand suggest that the limited cytokine response toS. aureusmay contribute to the well-known ability of the bacterium to establish chronic intramammary infection.

International audience; The objective of the current study was to evaluate the dynamics of infection and the immunological response to varying numbers of Escherichia coli injected into the mammary glands of primiparous cows during the periparturient period. Primiparous cows have been shown to be more resistant to intramammary E. coli challenge, and an increase of the inoculum dose by 2 log10 units induced a more rapid clinical response and clearance of the organisms. Recognition of lipopolysaccharide (LPS) is a key event in the innate immunity response to gram-negative infection and is mediated by the accessory molecules CD14 and LPS-binding protein (LBP). Primiparous cows were inoculated with 1 x 10(4) (Group A; n=8) or 1 x 10(6) (Group B; n=8) cfu E. coli P4:O32 in their 2 left quarters during the periparturient period. Clinical examination and analysis of blood and milk parameters, including IL-8, complement fragment 5a (C5a), LBP, and soluble CD14 (sCD14), were performed from d -4 to d +3 relative to infection. Primiparous cows in Group B initiated a more rapid clinical response following intramammary infection (IMI), resulting in typical clinical signs and changes in blood and milk parameters approximately 3 h earlier compared with primiparous cows in Group A. Based on average milk production in the noninfected quarters on d +2 postinoculation, all heifers reacted as moderate responders. Distinct differences in the kinetic patterns of rectal temperature, somatic cell count (SCC), IL-8, C5a, LBP, and sCD14 were observed between both groups during the early phase of inflammation. Both C5a and IL-8 increased before cellular influx into the infected glands, followed by increases in sCD14 and LBP. In conclusion, primiparous cows are able to clear an intramammary E. coli infection efficiently. Moreover, increasing the inoculum dose induces a more rapid inflammatory reaction, mainly because of early activation of the innate host immune response.

International audience; The mammary gland of dairy cows, which is prone to infection by various bacteria, mobilizes local and systemic immune defenses to cope with pathogens. The complement system plays an important part in the innate immunity against microorganisms through its bactericidal, opsonic, and phlogistic functions. The amount of the complement in the milk of healthy glands of dairy cows is low. Moreover, the classical pathway of activation is not functional because of a shortage in C1q. By contrast, the alternative pathway is active, deposits C3b and C3bi on bacteria, and generates amounts of C5a which are highly variable among cows. A slight inhibition of the bactericidal/hemolytic activities, of the deposition of C1q on bacteria, and of the phlogistic activity of C5a makes milk a rather anti-inflammatory fluid. The inhibitory activity does not involve C3b/ C3bi deposition on bacteria, nor the generation of C5a by the alternative pathway. When inflammation develops, the blood-derived complement components overcome the inhibitions and complement-dependent bactericidal, opsonic and phlogistic activities may be high in milk. Further research is necessary to evaluate the contribution of C5a to the recruitment of leukocytes in the mammary gland, and to specify the links between the complement system and the response of resident cells (leukocytes and mammary epithelial cells) to infection stimulus. This will help to define the contribution of the complement system to resistance against mastitis, and could help to differentiate animals more or less resistant to this frequent and costly disease.

Among the toxins that Staphylococcus aureus is able to secrete, bi-component toxins named leukotoxins target specifically leukocytes, mainly phagocytic cells. Isolates from cows, goats and ewes with mastitis were selected on the basis of the presence or not of the genes encoding the recently described LukM/LukF′-PV leukotoxin. Of the 128 isolates tested, 126 had moderate to high leukotoxic activity to bovine polymorphonuclear cells (PMN). The supernatants of lukM -positive isolates were much more leukotoxic than the supernatants of lukM -negative isolates: mean leukotoxic titers were 122 versus 20 and 581 versus 26 for isolates of bovine and caprine origin, respectively. Among lukM / lukF ′- PV positive isolates, those of caprine and ovine origins were more leukotoxic than were isolates of bovine origin ( P < 0.01). The two most abundant proteins in the culture supernatant of a highly toxic isolate were purified and identified as the two components of LukM (LukM and LukF′-PV) on the basis of their molecular mass, N-terminal amino acid sequence, and high synergistic activity. LukM/LukF′-PV induced the flattening of bovine PMN at a concentration as low as 3.6 ng/ml (0.1 nM). A higher concentration (18 ng/ml) was necessary to produce the same effect on caprine or ovine PMN. Affinity-purified antibodies to LukM or to LukF′-PV neutralized the leukotoxic effect of all the culture supernatants. They neutralized with the same efficiency the toxic activity of supernatants from lukM / lukF ′-PV positive or negative isolates. These results establish that LukM/LukF′-PV is very active on PMN of ruminants and suggest that this leukotoxin could be the most active leukotoxin produced by mastitis isolates. They prompt further studies to delineate the contribution of LukM/LukF′-PV to the pathogenesis of mastitis in ruminants and the protective effect of antibodies to this leukotoxin.

International audience; The leucocytes present in normalmilk are not very efficient in preventing infection, because very small numbers of bacteria are able to induce infection experimentally. The mobilization of phagocytes from the blood to milk appears crucial in coping with the expansion of the bacterial population in the mammary gland. Important parameters for the outcome of mammary infections are the bactericidal efficiency of neutrophils and the antiphagocytic and cytotoxic properties of the invading bacteria, but several studies have shown that the promptness and the magnitude of the initial recruitment of neutrophils by the infected mammary gland have a profound influence on the severity and the outcome of mastitis. This is an incentive for studying the mechanisms behind the mobilization of neutrophils to the mammary gland. Although milk macrophages may play a role in the triggering of the inflammatory response, studies on several responses to infections at various epithelium sites strongly suggest that epithelial cells are capable of responding to bacterial intrusion and play a major part in the initiation of inflammation. A better knowledge of the effector cells and of the mediators involved in the mobilization of neutrophils could help in devising strategies to modulate this important determinant of milk quality and udder defense.

Phagocytosis of bacteria by bovine polymorphonuclear neutrophils (PMN) has long been regarded as essential for host defense against mastitis infection. Complement-mediated opsonisation by complement component 3 (C3) binding is an important component of the innate immune system. We investigated the role of milk complement as an opsonin and its involvement in the phagocytosis and killing of Staphylococcus aureus isolates from cases of bovine mastitis by bovine blood PMN. We show that deposition of milk C3 component occurred on six different isolates of S. aureus and that the alternative pathway was the sole complement pathway operating in milk of uninflamed mammary gland. This deposition was shown to occur at the same location as the capsule, but not on capsular antigen. Milk complement enhanced the chemiluminescence response of PMN induced by S. aureus. Nevertheless, the association of S. aureus to cells and the overall killing of bacteria by bovine PMN were not affected by the presence of milk complement. Therefore, as all milk samples contained antibodies to capsular polysaccharide type 5 and to other surface antigens, it is likely that milk antibodies were responsible for these two phagocytic events. Results of this study suggest that the deposition of milk complement components on the surface of S. aureus does not contribute to the defence of the mammary gland against S. aureus.

The phagocytosis of Staphylococcus aureus by bovine polymorphonuclear neutrophils (PMN) requires the presence of antibodies. Among the major isotypes of bovine antibodies, IgG2 and IgM are considered opsonic for bovine PMN. However, the role of purified bovine secretory IgA (sIgA) as an opsonin has not been assessed. In the present study, IgG2 were obtained from serum and sIgA, IgG1, and IgM were purified from the colostrums of three cows intramammarily immunized with heat-killed Staphylococcus aureus. The Ig preparations were assayed for specific antibodies, and the opsonic capacity of every isotype was investigated. Despite the presence of antibodies, we observed no distinct chemiluminescence response of PMN stimulated with sIgA- or IgG1-opsonized S. aureus, whereas IgM or IgG2 bound to bacteria induced a marked chemiluminescence response. Moreover, the counting of internalized bacteria per PMN after phagocytosis revealed a low uptake of S. aureus opsonized with sIgA or IgG1, in contrast to IgM or IgG2, which triggered efficient ingestion of bacteria. Priming of neutrophils by TNF-alpha, IFN-gamma, or C5adesArg did not promote an oxidative burst or uptake of sIgA-opsonized S. aureus to a greater extent than with IgG1-opsonized bacteria. Furthermore, analysis of uningested bacteria by flow cytometry after incubation with PMN showed a preferential uptake of IgM-opsonized S. aureus by PMN and only few sIgA-positive stained bacteria were PMN-associated. These experiments indicate that sIgA, like IgG1 and unlike IgM or IgG2, could not be considered as a major opsonin for phagocytosis of S. aureus by bovine blood PMN.

The contribution of the alternative pathway of complement activation to the capacity of normal milk to deposit C3 fragments on bacteria was tested by attempting to block C3 deposition with antibodies to the alternative pathway component factor B (fB). Factor B was purified and antibodies of the IgY class, which does not activate mammalian complement, were obtained from the egg yolk of immunized laying hens. These antibodies specifically inhibited the deposition of C3. This inhibition and the absence of deposition of C4 demonstrated that C3 deposition in normal milk resulted from the activation of the alternative pathway. Antibodies raised in rabbit were used to develop an ELISA for measuring fB concentrations in milk. The mean concentration of fB was 2·06 μg/ml (±0·18, SEM), 0·57% of the mean value found in serum (360 μg/ml). This proportion was comparable to that of serum albumin (0·63% of serum value) but less than the proportion of C3 in milk (2·71%). Nevertheless, fB was apparently not a limiting factor for the functioning of the alternative pathway, since addition of purified fB to normal milk did not improve C3 deposition. In serum, mild heat-treatment (56 °C for 3 min or 50 °C for 45 min) blocked the alternative pathway and destroyed fB, as shown by loss of antigenicity in ELISA. In milk, mild heat-treatment did not abrogate C3 deposition, and fB was protected, retaining its functionality and antigenicity. Heating at 56 °C for at least 45 min was necessary to completely inhibit C3 deposition in normal milk.

Phagocytic and bactericidal activity of polymorphonuclear neutrophil leukocytes (PMN) isolated from blood and milk, against Staphylococcus aureus, was compared between groups of six healthy dairy cows in early, mid- and late lactation using a bacteriological assay. PMN were isolated from blood with a high degree of purity, but the cells isolated from milk contained variable amounts of macrophages (MΦ) and lymphocytes (L). The results were therefore calculated using the percentage PMN in order to evaluate phagocytosis and killing by PMN only. Blood PMN phagocytosed 82% Staph. aureus and milk PMN 43% on average and there was no significant difference between the different stages of lactation. The bactericidal activity of blood PMN against Staph. aureus was 36±8% in early lactation (significantly different from mid lactation, PStaph. aureus in early lactation (significantly different from mid lactation, PStaph. aureus in late lactation. The ratio of the bactericidal activity of milk to blood PMN was 0·08, 0·43 and 0·22 in early, mid- and late lactation, respectively. In addition to the decreased function, the number of cells in milk (somatic cell count, SCC) was also 60% lower in early lactation than in mid lactation cows (PStaph. aureus and a decreased number of milk-resident PMN in dairy cows at the onset of lactation.

Staphylococcus aureus is a major pathogen in bovine intramammary infections of subclinical and chronic nature. Persistent infection with S. aureus has been postulated to be associated with an impaired immune response. This study was designed to define changes in peripheral blood and milk cell subpopulations during chronic S. aureus infection. The expression of specific antigens on the surface of lymphocytes and neutrophils was studied by flow cytometry. Cytokines and cytokine transcripts elaborated by the milk-derived cells were also investigated, using ELISA and reverse transcription polymerase chain reaction, respectively. The results indicated that cell subpopulations in blood from infected cows were not modified. In contrast, changes occurred in infected milk: neutrophils were the main cell population, but they were not in a highly activated state; the CD8+ T-lymphocytes were mainly recruited compared with the CD4+ T-lymphocytes, suggesting that CD8+ T-lymphocytes play an important role in chronic S. aureus infection. Also, the proportion of the B-lymphocytes among the total lymphocyte population was increased, suggesting that a humoral response developed, and no change was observed in the gammadelta subset. No cytokine mRNA was found in milk cells from uninfected mammary glands. In contrast, interleukin (IL)-1alpha, IL-1beta, IL-6 and tumor necrosis factor alpha pro-inflammatory cytokine and IL-10 and IL-12 regulatory cytokine mRNA were synthesized in cells derived from infected mammary glands, whereas no IL-2 nor IL-4 mRNA were found. Therefore, cells present in milk during chronic S. aureus infection were activated, but did not reveal any polarization of the immune response.