T helper 17-associated cytokines are produced during antigen-specific inflammation in the mammary gland.

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.

Purified Staphylococcus aureus leukotoxin LukM/F' does not trigger inflammation in the bovine mammary gland.

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-α, IL-1ß 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.

Muramyl dipeptide synergizes with Staphylococcus aureus lipoteichoic acid to recruit neutrophils in the mammary gland and to stimulate mammary epithelial cells.

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-1ß 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.

Binding of the Staphylococcus aureus leucotoxin LukM to its leucocyte targets.

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 (HlgA, HlgC, 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.

Staphylococcus aureus lipoteichoic acid triggers inflammation in the lactating bovine mammary gland.

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 100 microg/quarter, and a subclinical inflammatory response at 10 microg/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 (100 microg). The pro-inflammatory cytokine IL-1beta was induced in milk, but there was very little if any TNF-alpha and no IFN-gamma. 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. aureus LTA 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.

The chemokine CXCL3 is responsible for the constitutive chemotactic activity of bovine milk for neutrophils.

Bovine milk is known to exert a potent chemotactic activity on neutrophils, but the responsible agent has not been identified. The objective of the study was to characterize the main biochemical component responsible for this chemotactic activity. A neutrophil shape change assay was used to locate active milk fractions separated by chromatography. A single protein was isolated and identified by amino acid sequencing and mass spectrometry as CXCL3. Recombinant bovine chemokines and specific antibodies were used to show that normal milk contains active concentrations of CXCL1 (1-5ng/ml) and CXCL3 (100-500ng/ml), whereas CXCL2 and CXCL8/IL-8 were not detected. Depletion experiments with antibodies showed that CXCL3 was the main chemotaxin for neutrophils in normal (non-mastitic) milk. The chemokine CXCL3 was located by immunohistochemistry in mammary epithelial cells, and abundant mRNA was found in uninflamed mammary tissue, suggesting constitutive secretion by the lactating mammary epithelium. These results indicate that CXCL3/GRO-gamma is the major chemotactic factor for neutrophils in bovine milk in the absence of inflammation, and that it is secreted constitutively in milk by mammary epithelial cells. This finding prompts the question of the biological significance of permanent high concentrations of a CXC chemokine in milk.

Evaluation of tandem repeats for MLVA typing of Streptococcus uberis isolated from bovine mastitis.

BACKGROUND: Streptococcus uberis is a common cause of bovine mastitis and recommended control measures, based on improved milking practice, teat dipping and antibiotic treatment at drying-off, are poorly efficient against this environmental pathogen. A simple and efficient typing method would be helpful in identifying S.uberis sources, virulent strains and cow to cow transmission. The potential of MLVA (Multiple Loci VNTR Analysis; VNTR, Variable Number of Tandem Repeats) for S. uberis mastitis isolates genotyping was investigated. RESULTS: The genomic sequence of Streptococcus uberis (strain 0104J) was analyzed for potential variable number tandem repeats (VNTRs). Twenty-five tandem repeats were identified and amplified by PCR with DNA samples from 24 S. uberis strains. A set of seven TRs were found to be polymorphic and used for MLVA typing of 88 S. uberis isolates. A total of 82 MLVA types were obtained with 22 types among 26 strains isolated from the milk of mastitic cows belonging to our experimental herd, and 61 types for 62 epidemiologically unrelated strains, i.e. collected in different herds and areas. CONCLUSION: The MLVA method can be applied to S. uberis genotyping and constitutes an interesting complement to existing typing methods. This method, which is easy to perform, low cost and can be used in routine, could facilitate investigations of the epidemiology of S. uberis mastitis in dairy cows.

Differentiation of bovine Staphylococcus aureus isolates by use of polymorphic tandem repeat typing.

Staphylococcus aureus is a common cause of bovine mastitis. A simple and efficient typing method would be helpful in understanding S. aureus sources and spread. Ninety-six S. aureus strains, isolated between 1961 and 2003 from the milk of 90 dairy cows belonging to 75 French herds, were subjected to multiple-locus variable-number tandem repeats analysis (MLVA) by PCR. The conjunction of clfA, clfB, SAV1078 and fnb gene tandem repeats (TRs) enabled the definition of 61 types. When coa, spa, sdrC, sdrD and sspA TRs were used individually as additional markers, 63, 68, 67, 65 and 67 types were defined, respectively, versus 77 types when they were all included in the method. These additional TRs did not improve the differentiation of isolates collected in the same farm. The MLVA procedure using the tandem repeats embedded in clfA, clfB, SAV1078 and fnb loci as a basic combination at the herd level or associated with other TRs such as spa, sdrC, sdrD, sspA and coa can be a valuable tool for bovine S. aureus epidemiological studies.