Publications by authors named "Pascal Rainard"

50 Publications

Shielding Effect of Escherichia coli O-Antigen Polysaccharide on J5-Induced Cross-Reactive Antibodies.

mSphere 2021 01 27;6(1). Epub 2021 Jan 27.

INRAE, Université de Tours, UMR ISP, Nouzilly, France

is the leading cause of severe mastitis in dairy farms. As mastitis is refractory to the hygienic control measures adapted to contagious mastitis, efficient vaccines are in demand. Existing mastitis vaccines, based on the use of killed rough J5 as the antigen, aim at inducing phagocytosis by neutrophils. We assessed the binding of J5-induced antibodies to isogenic rough and smooth strains along with a panel of mastitis-associated Analysis by enzyme-linked immunosorbent assay revealed that antibodies to OmpA or killed J5 bind readily to rough but poorly to smooth strains. Flow cytometry analysis indicated that immunization with J5 induced antibodies that cross-reacted with rough strains but with only a small subpopulation of smooth strains. We identified type 1 fimbriae as the target of most antibodies cross-reacting with the smooth strains. These results suggest that the O-polysaccharide of lipopolysaccharide shields the outer membrane antigens and that only fiber antigens protruding at the bacterial surface can elicit antibodies reacting with mastitis-associated We evaluated J5-induced antibodies in an opsonophagocytic killing assay with bovine neutrophils. J5 immune serum was not more efficient than preimmune serum, showing that immunization did not improve on the already high efficiency of naturally acquired antibodies to In conclusion, it is unlikely that the efficiency of J5 vaccines is related to the induction of opsonic antibodies. Consequently, other research directions, such as cell-mediated immunity, should be explored to improve mastitis vaccines. Despite intensive research, mastitis remains an important disease in dairy cattle with a significant impact on animal welfare, use of antibiotics, and, in the end, the economy of dairy farms. Although vaccines available so far have shown limited efficacy against coliform mastitis, vaccination is considered one of the measures that could limit the consequences of mastitis. One reason for the lack of efficiency of current vaccines likely stems from the current evaluation of vaccines that relies mostly on measuring antibody production against vaccine antigens. This report clearly shows that vaccine-induced antibodies fail to bind to most mastitis-associated strains because of the presence of an O-antigen and, thus, do not allow for improved phagocytosis of pathogens. As a consequence, this report calls for revised criteria for the evaluation of vaccines and suggests that cell-mediated immunity should be targeted by new vaccinal strategies. More generally, these results could be extended to other vaccine development strategies targeting coliform bacteria.
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http://dx.doi.org/10.1128/mSphere.01227-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7885324PMC
January 2021

Th17-related mammary immunity, but not a high systemic Th1 immune response is associated with protection against E. coli mastitis.

NPJ Vaccines 2020 Nov 24;5(1):108. Epub 2020 Nov 24.

IHAP, Université de Toulouse, ENVT, INRAE, 31076, Toulouse, France.

Vaccination against bovine mastitis lags behind despite high demand from the dairy industry and margin for efficacy improvement. We previously compared two immunization protocols against E. coli using either only the intramuscular route or a combination of intramuscular and mammary ductal routes, also known as 'prime and pull' strategy. A homologous mammary challenge during the memory phase showed that immunization favorably modified the mastitis course, notably in locally immunized cows in comparison to intramuscular and control adjuvant-only groups. Here, we performed whole-blood profiling through RNA-seq transcriptome and plasma cytokine 15-plex analyses at time points of the E. coli mastitis that showed significant clinical and laboratory differences among the groups. Diminished production of inflammatory cytokines and increased IFNγ were detected in the blood of immunized cows, where a T lymphocyte activation profile was evidenced at 12-h post infection. Acute phase neutropenia was less severe in these cows, and pathways related to neutrophil diapedesis and monocyte activation were also present. Furthermore, three intramammary-immunized cows showing faster healing and shorter mastitis duration had gene profiles that differed from their counterparts, but without any clue for the mastitis susceptibility difference. Inasmuch, when gene expression of CD4 T cells was assessed in mammary tissue, enrichment of IL-17-associated pathways was identified in the quarters of intramammary-immunized cows not only after challenge but also in the control quarters that were not infected. These findings indicate that local immunization mobilizes protective mechanisms that rely on the settlement of type 3 immunity-related CD4 T cells prior to infection.
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http://dx.doi.org/10.1038/s41541-020-00258-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686320PMC
November 2020

Type 3 immunity: a perspective for the defense of the mammary gland against infections.

Vet Res 2020 Oct 15;51(1):129. Epub 2020 Oct 15.

IHAP, Université de Toulouse, INRAE, ENVT, Toulouse, France.

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.
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http://dx.doi.org/10.1186/s13567-020-00852-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7559147PMC
October 2020

Expansion, isolation and first characterization of bovine Th17 lymphocytes.

Sci Rep 2019 11 6;9(1):16115. Epub 2019 Nov 6.

ISP, INRA, Université de Tours, UMR1282, Nouzilly, France.

Interleukin 17A-producing T helper cells (Th17) are CD4+ T cells that are crucial to immunity to extracellular bacteria. The roles of these cells in the bovine species are poorly defined, because the characterization of bovine Th17 cells lags behind for want of straightforward cultivation and isolation procedures. We have developed procedures to differentiate, expand, and isolate bovine Th17 cells from circulating CD4+ T cells of adult cows. Using polyclonal stimulation with antibodies to CD3 and CD28, we expanded IL-17A-positive CD4+ T cells in a serum-free cell culture medium supplemented with TGF-β1, IL-6 and IL-2. Populations of CD4+ T cells producing IL-17A or IFN-γ or both cytokines were obtained. Isolation of IL-17A-secreting CD4+ T cells was performed by labelling surface IL-17A, followed by flow cytometry cell sorting. The sorted Th17 cells were restimulated and could be expanded for several weeks. These cells were further characterized by cytokine profiling at transcriptomic and protein levels. They produced high amounts of IL-17A and IL-17F, and moderate amounts of IL-22 and IFN-γ. The techniques developed will be useful to characterize the phenotypic and functional properties of bovine Th17 cells.
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http://dx.doi.org/10.1038/s41598-019-52562-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6834651PMC
November 2019

A reply to the comments on "Control of bovine mastitis in the 21st century: Immunize of tolerize?"

Authors:
Pascal Rainard

Res Vet Sci 2019 12 16;127:103-104. Epub 2019 Oct 16.

ISP, INRA, Université de Tours, UMR1282, Nouzilly, France. Electronic address:

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http://dx.doi.org/10.1016/j.rvsc.2019.09.014DOI Listing
December 2019

A Critical Appraisal of Probiotics for Mastitis Control.

Front Vet Sci 2018 10;5:251. Epub 2018 Oct 10.

IHAP, Université de Toulouse, ENVT, INRA, UMR1225, Toulouse, France.

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 experiments and the trials. Preliminary experiments aimed essentially at exploring 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 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.
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http://dx.doi.org/10.3389/fvets.2018.00251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6191464PMC
October 2018

Sensing of Escherichia coli and LPS by mammary epithelial cells is modulated by O-antigen chain and CD14.

PLoS One 2018 24;13(8):e0202664. Epub 2018 Aug 24.

ISP UMR 1282, INRA, Université François Rabelais de Tours, Nouzilly, France.

Escherichia coli is one of the major pathogens causing mastitis in dairy cattle. Yet, the factors which mediate the ability for E. coli to develop in the bovine mammary gland remain poorly elucidated. In a mouse model, infections induced by the reference mastitis E. coli P4 showed a strong colonisation of the mammary gland, while this strain had a low stimulating power on cells of the PS bovine mammary epithelial cell line. In order to understand if such a reduced response contributes to the severity of infection, a library of random mutants of P4 strain was screened to identify mutants inducing stronger response of PS cells. Among hyper-stimulating mutants, six were altered in genes involved in biosynthesis of lipopolysaccharide (LPS) and had lost their O-polysaccharide region, suggesting that the presence of O-antigen impairs the response of PS cells to LPS. Using purified smooth (S) and rough (R) fractions of LPS, we showed that the R-LPS fraction induced a stronger response from PS cells than the smooth LPS fraction. Biological activity of the S-LPS fraction could be restored by the addition of recombinant bovine CD14 (rbCD14), indicating a crucial role of CD14 in the recognition of S-LPS by Mammary Epithelial Cells (MEC). When S-LPS and R-LPS were injected in udder quarters of healthy lactating cows, an inflammation developed in all infused quarters, but the S-LPS induced a more intense pro-inflammatory response, possibly in relation to sizeable concentrations of CD14 in milk. Altogether, our results demonstrate that the O-antigen modulates the pro-inflammatory response of MEC to LPS, that S-LPS and R-LPS trigger different responses of MEC and that these responses depend on the presence of CD14.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0202664PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6108492PMC
February 2019

Host factors determine the evolution of infection with Staphylococcus aureus to gangrenous mastitis in goats.

Vet Res 2018 07 25;49(1):72. Epub 2018 Jul 25.

GABI, INRA, UMR 1313, Université Paris Saclay, 78350, Jouy-en-Josas, France.

Staphylococcus aureus is the major cause of very severe mastitis of dairy goats. The initial objective of our study was to fine-tune an experimental model of infection of the goat mammary gland with two strains of S. aureus and two lines of goats (low and high somatic cell score lines). Following the challenge, the 10 infected goats divided in two clear-cut severity groups, independently of the S. aureus strain and the goat line. Five goats developed very severe mastitis (of which four were gangrenous) characterized by uncontrolled infection (UI group), whereas the other five kept the infection under control (CI group). The outcome of the infection was determined by 18 h post-infection (hpi), as heralded by the bacterial milk concentration at 18 hpi: more than 10/mL in the UI group, about 10/mL in the CI group. Leukocyte recruitment and composition did not differ between the groups, but the phagocytic killing at 18 hpi efficiency did. Contributing factors involved milk concentrations of α-toxin and LukMF' leukotoxin, but not early expression of the genes encoding the pentraxin PTX3, the cytokines IL-1α and IL-1β, and the chemokines IL-8 and CCL5. Concentrations of TNF-α, IFN-γ, IL-17A, and IL-22 rose sharply in the milk of UI goats when infection was out of control. The results indicate that defenses mobilized by the mammary gland at an early stage of infection were essential to prevent staphylococci from reaching critical concentrations. Staphylococcal exotoxin production appeared to be a consequent event inducing the evolution to gangrenous mastitis.
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http://dx.doi.org/10.1186/s13567-018-0564-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6060506PMC
July 2018

Postgenomics Characterization of an Essential Genetic Determinant of Mammary Pathogenic .

mBio 2018 04 3;9(2). Epub 2018 Apr 3.

IB3, Heriot Watt University, Edinburgh, United Kingdom.

are major bacterial pathogens causing bovine mastitis, a disease of great economic impact on dairy production worldwide. This work aimed to study the virulence determinants of mammary pathogenic (MPEC). By whole-genome sequencing analysis of 40 MPEC and 22 environmental ("dairy-farm" [DFEC]) strains, we found that only the locus () for ferric dicitrate uptake was present in the core genome of MPEC and that it was absent in DFEC genomes ( < 0.05). Expression of the FecA receptor in the outer membrane was shown to be citrate dependent by mass spectrometry. FecA was overexpressed when bacteria were grown in milk. Transcription of the gene and of the inner membrane transport component gene was upregulated in bacteria recovered from experimental intramammary infection. The presence of the system was shown to affect the ability of to grow in milk. While the rate of growth in milk of -positive () DFEC was similar to that of MPEC, it was significantly lower in DFEC lacking Furthermore, deletion of reduced the rate of growth in milk of MPEC strain P4, whereas -transformed non-mammary gland-pathogenic DFEC strain K71 gained the phenotype of the level of growth in milk observed in MPEC. The role of in intramammary pathogenicity was investigated in cows, with results showing that an MPEC P4 mutant lacking lost its ability to induce mastitis, whereas the DFEC K71 mutant was able to trigger intramammary inflammation. For the first time, a single molecular locus was shown to be crucial in MPEC pathogenicity. Bovine mastitis is the major infectious disease in dairy cows and the leading cause of economic loss to the global dairy industry, directly contributing to the price of dairy products on supermarket shelves and the financial hardships suffered by dairy farmers. Mastitis is also the leading reason for the use of antibiotics in dairy farms. Good farm management practices in many countries have dramatically reduced the incidence of contagious mastitis; however, the problems associated with the incidence of environmental mastitis caused by bacteria such as have proven intractable. bacteria cause acute mastitis, which affects the health and welfare of cows and in extreme cases may be fatal. Here we show for the first time that the pathogenicity of causing mastitis in cows is highly dependent on the ferric citrate uptake system that allows the bacterium to capture iron from citrate. The Fec system is highly expressed during infection in the bovine udder and is ubiquitous in and necessary for the bacteria that cause mammary infections in cattle. These results have far-reaching implications, raising the possibility that mastitis may be controllable by targeting this system.
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http://dx.doi.org/10.1128/mBio.00423-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5885034PMC
April 2018

Cellular and humoral immune response to recombinant Escherichia coli OmpA in cows.

PLoS One 2017 31;12(10):e0187369. Epub 2017 Oct 31.

Infectiologie et Santé Publique, UMR 1282, INRA, Université Tours, Nouzilly, France.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0187369PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5663511PMC
November 2017

Behavioral and patho-physiological response as possible signs of pain in dairy cows during Escherichia coli mastitis: A pilot study.

J Dairy Sci 2017 Oct 26;100(10):8385-8397. Epub 2017 Jul 26.

Université de Toulouse, Ecole Nationale Vétérinaire de Toulouse (ENVT), INRA, Interactions Hôtes-Agents Pathogènes (IHAP), F-31076 Toulouse, France.

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 µg/mL) concentrations, and rumen temperature was increased (40.3°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 µg/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.
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http://dx.doi.org/10.3168/jds.2017-12796DOI Listing
October 2017

Escherichia coli mastitis strains: In vitro phenotypes and severity of infection in vivo.

PLoS One 2017 20;12(7):e0178285. Epub 2017 Jul 20.

ISP, INRA, Université François Rabelais de Tours, UMR 1282, Nouzilly, France.

Mastitis remains a major infection of dairy cows and an important issue for dairy farmers and the dairy industry, in particular infections due to Escherichia coli strains. So far, properties specific to E. coli causing mastitis remain ill defined. In an attempt to better understand the properties required for E. coli to trigger mastitis, we used a range of in vitro assays to phenotypically characterize four E. coli strains, including the prototypical E. coli mastitis strain P4, possessing different relative abilities to cause mastitis in a mouse model. Our results indicate that a certain level of serum resistance might be required for colonization of the mammary gland. Resistance to neutrophil killing is also likely to contribute to a slower clearance of bacteria and higher chances to colonize the udder. In addition, we show that the four different strains do induce a pro-inflammatory response by mammary epithelial cells but with different intensities. Interestingly, the prototypical mastitis strain P4 actually induces the less intense response while it is responsible for the most severe infections in vivo. Altogether, our results suggest that different strategies can be used by E. coli strains to colonize the mammary gland and cause mastitis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0178285PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5519002PMC
September 2017

Local immunization impacts the response of dairy cows to Escherichia coli mastitis.

Sci Rep 2017 06 13;7(1):3441. Epub 2017 Jun 13.

ISP, INRA, Université de Tours, UMR1282, Nouzilly, France.

Current vaccines to Escherichia coli mastitis have shown some albeit limited efficacy. Their mode of action has not been documented, and immune responses protecting the mammary gland against E. coli are not completely understood. To improve our knowledge of mammary gland immune protection, cows immunized either intramuscularly or intramammarily with the E. coli P4 were submitted to a homologous mastitis challenge. A third group of mock-immunized cows serve as challenge controls. Local immunization modified favorably the course of infection, by improving bacterial clearance while limiting inflammation. Systemic clinical signs and reduction in milk secretion were also contained. This occurred with a modification of the cytokine profile, such as an increase in IFN-γ and a reduction in TNF-α concentrations in milk. Concentrations of IL-17A and IL-22 increased in milk at the onset of the inflammatory response and remained high up to the elimination of bacteria, but concentrations did not differ between groups. Accelerated bacteriological cure was not linked to an increase in the initial efficiency of phagocytosis in milk. Results support the idea that antibodies did not play a major role in the improvement, and that cell-mediated immunity is the key to understanding E. coli vaccine-induced protection of the mammary gland.
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http://dx.doi.org/10.1038/s41598-017-03724-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5469773PMC
June 2017

Mammary microbiota of dairy ruminants: fact or fiction?

Authors:
Pascal Rainard

Vet Res 2017 04 17;48(1):25. Epub 2017 Apr 17.

ISP, INRA, Université de Tours, UMR1282, 37380, Nouzilly, France.

Explorations of how the complex microbial communities that inhabit different body sites might contribute to health and disease have prompted research on the ways the harmonious relationship between a host and its microbiota could be used to keep animals healthy in their production conditions. In particular, there is a growing interest in the bacterial signatures that can be found in the milk of healthy or mastitic dairy cows. The concept of sterility of the healthy mammary gland of dairy ruminants has been challenged by the results of studies using bacterial DNA-based methodology. The newly obtained data have led to the concept of the intramammary microbiota composed of a complex community of diverse bacteria. Accordingly, mammary gland infections are not mere infections by a bacterial pathogen, but the consequence of mammary dysbiosis. This article develops the logical implications of this paradigm shift and shows how this concept is incompatible with current knowledge concerning the innate and adaptive immune system of the mammary gland of dairy ruminants. It also highlights how the concept of mammary microbiota clashes with results of experimental infections induced under controlled conditions or large field experiments that demonstrated the efficacy of the current mastitis control measures.
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http://dx.doi.org/10.1186/s13567-017-0429-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5392980PMC
April 2017

Permissiveness of bovine epithelial cells from lung, intestine, placenta and udder for infection with Coxiella burnetii.

Vet Res 2017 04 12;48(1):23. Epub 2017 Apr 12.

Institute of Molecular Pathogenesis, Friedrich-Loeffler-Institut (FLI), Naumburger Strasse 96a, 07743, Jena, Germany.

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.
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http://dx.doi.org/10.1186/s13567-017-0430-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389005PMC
April 2017

Innate and Adaptive Immunity Synergize to Trigger Inflammation in the Mammary Gland.

PLoS One 2016 21;11(4):e0154172. Epub 2016 Apr 21.

ISP, INRA, Université Tours, Nouzilly, France.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0154172PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4839698PMC
April 2017

Staphylococcus aureus Phenol-Soluble Modulins Impair Interleukin Expression in Bovine Mammary Epithelial Cells.

Infect Immun 2016 06 24;84(6):1682-1692. Epub 2016 May 24.

INRA, UMR1253 STLO, Rennes, France

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.
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http://dx.doi.org/10.1128/IAI.01330-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4907149PMC
June 2016

IL-17A Is an Important Effector of the Immune Response of the Mammary Gland to Escherichia coli Infection.

J Immunol 2016 Jan 18;196(2):803-12. Epub 2015 Dec 18.

UMR1282 Infectiologie et Santé Publique, Institut National de la Recherche Agronomique, F-37380 Nouzilly, France; and UMR1282 Infectiologie et Santé Publique, Université François-Rabelais de Tours, F-37000 Tours, France

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.
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http://dx.doi.org/10.4049/jimmunol.1500705DOI Listing
January 2016

Antigen-Specific Mammary Inflammation Depends on the Production of IL-17A and IFN-γ by Bovine CD4+ T Lymphocytes.

PLoS One 2015 16;10(9):e0137755. Epub 2015 Sep 16.

INP, ENVT, Université de Toulouse, Toulouse, France; UMR1225, Interactions Hôte Agents Pathogènes, INRA, Toulouse, France.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0137755PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4573518PMC
May 2016

Investigating the contribution of IL-17A and IL-17F to the host response during Escherichia coli mastitis.

Vet Res 2015 Jun 11;46:56. Epub 2015 Jun 11.

INRA, UMR1282, Infectiologie et Santé Publique, F-37380, Nouzilly, France.

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.
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http://dx.doi.org/10.1186/s13567-015-0201-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4462179PMC
June 2015

Location-specific expression of chemokines, TNF-α and S100 proteins in a teat explant model.

Innate Immun 2015 Apr 17;21(3):322-31. Epub 2014 Aug 17.

Clinic for Ruminants with Ambulatory and Herd Health Services at the Centre for Clinical Veterinary Medicine, Ludwig-Maximilians University Munich, Oberschleissheim, Germany

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.
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http://dx.doi.org/10.1177/1753425914539820DOI Listing
April 2015

Impact of intramammary treatment on gene expression profiles in bovine Escherichia coli mastitis.

PLoS One 2014 14;9(1):e85579. Epub 2014 Jan 14.

Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0085579PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3891811PMC
September 2014

Differential response of bovine mammary epithelial cells to Staphylococcus aureus or Escherichia coli agonists of the innate immune system.

Vet Res 2013 Jun 11;44:40. Epub 2013 Jun 11.

INRA, UMR1282, ISP, F 37380, Nouzilly, France.

Mastitis caused by Escherichia coli and Staphylococcus aureus is a major pathology of dairy cows. To better understand the differential response of the mammary gland to these two pathogens, we stimulated bovine mammary epithelial cells (bMEC) with either E. coli crude lipopolysaccharide (LPS) or with S. aureus culture supernatant (SaS) to compare the transcriptomic profiles of the initial bMEC response. By using HEK 293 reporter cells for pattern recognition receptors, the LPS preparation was found to stimulate TLR2 and TLR4 but not TLR5, Nod1 or Nod2, whereas SaS stimulated TLR2. Biochemical analysis revealed that lipoteichoic acid, protein A and α-hemolysin were all present in SaS, and bMEC were found to be responsive to each of these molecules. Transcriptome profiling revealed a core innate immune response partly shared by LPS and SaS. However, LPS induced expression of a significant higher number of genes and the fold changes were of greater magnitude than those induced by SaS. Microarray data analysis suggests that the activation pathways and the early chemokine and cytokine production preceded the defense and stress responses. A major differential response was the activation of the type I IFN pathway by LPS but not by SaS. The higher upregulation of chemokines (Cxcl10, Ccl2, Ccl5 and Ccl20) that target mononuclear leucocytes by LPS than by SaS is likely to be related to the differential activation of the type I IFN pathway, and could induce a different profile of the initial recruitment of leucocytes. The MEC responses to the two stimuli were different, as LPS was associated with NF-κB and Fas signaling pathways, whereas SaS was associated with AP-1 and IL-17A signaling pathways. It is noteworthy that at the protein level secretion of TNF-α and IL-1β was not induced by either stimulus. These results suggest that the response of MEC to diffusible stimuli from E. coli and S. aureus contributes to the onset of the response with differential leucocyte recruitment and distinct inflammatory and innate immune reactions of the mammary gland to infection.
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http://dx.doi.org/10.1186/1297-9716-44-40DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3686618PMC
June 2013

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

PLoS One 2013 16;8(5):e63471. Epub 2013 May 16.

Infectiology and Public Health Research Unit, Institut National de la Recherche Agronomique, Nouzilly, France.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0063471PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3656053PMC
December 2013

Genetic susceptibility to S. aureus mastitis in sheep: differential expression of mammary epithelial cells in response to live bacteria or supernatant.

Physiol Genomics 2012 Apr 14;44(7):403-16. Epub 2012 Feb 14.

Université de Toulouse, Institut National Polytechnique (INP), École Nationale Vétérinaire de Toulouse (ENVT), Unité Mixte de Recherche (UMR)1225, Interactions Hôtes - Agents Pathogènes (IHAP), Toulouse, France.

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 response-related 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.
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http://dx.doi.org/10.1152/physiolgenomics.00155.2011DOI Listing
April 2012

Repertoire of Escherichia coli agonists sensed by innate immunity receptors of the bovine udder and mammary epithelial cells.

Vet Res 2012 Feb 13;43:14. Epub 2012 Feb 13.

INRA, UMR 1282 Infectiologie et Santé Publique, F-37380 Nouzilly, France.

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.
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http://dx.doi.org/10.1186/1297-9716-43-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305352PMC
February 2012

Staphylococcal-associated molecular patterns enhance expression of immune defense genes induced by IL-17 in mammary epithelial cells.

Cytokine 2011 Dec 17;56(3):749-59. Epub 2011 Oct 17.

INRA, UR1282 Infectiologie Animale et Santé Publique (IASP), F-37380 Nouzilly, France.

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-α. 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-α and IL-1β 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.
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http://dx.doi.org/10.1016/j.cyto.2011.09.020DOI Listing
December 2011

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

Microb Pathog 2011 Dec 22;51(6):396-401. Epub 2011 Sep 22.

INRA, UR 1282 Infectiologie Animale et Santé Publique, IASP Bat. 311, F-37380 Nouzilly, France.

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-1β 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.
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http://dx.doi.org/10.1016/j.micpath.2011.09.005DOI Listing
December 2011

Issues and special features of animal health research.

Vet Res 2011 Aug 24;42:96. Epub 2011 Aug 24.

INRA, UR346 Epidémiologie animale, 63122 Saint Genès Champanelle, France.

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.
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http://dx.doi.org/10.1186/1297-9716-42-96DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3170600PMC
August 2011

Genomic and proteomic characterization of Staphylococcus aureus mastitis isolates of bovine origin.

Proteomics 2011 Jun 18;11(12):2491-502. Epub 2011 May 18.

Institute for Microbiology, University of Greifswald, Greifswald, Germany.

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.
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http://dx.doi.org/10.1002/pmic.201000698DOI Listing
June 2011