Bernard Poutrel

Differential induction of complement fragment C5a and inflammatory cytokines during intramammary infections with Escherichia coli and Staphylococcus aureus.

ABSTRACT The prompt recruitment of neutrophils to the site of infection is essential for the defense of the bovine mammary gland against invading pathogens and is determinant for the outcome of the infection.Escherichia coli is known to induce clinical mastitis, characterized by an intense neutrophil recruitment leading to the eradication of the bacteria, whereas Staphylococcus aureusinduces subclinical mastitis accompanied by a moderate neutrophil recruitment and the establishment of chronic mastitis. To elicit the neutrophil recruitment into the udder, inflammatory mediators must be produced after recognition of the invading pathogen. To our knowledge, those mediators have never been studied during S. aureusmastitis, although understanding of the neutrophil recruitment mechanisms could allow a better understanding of the differences in the pathogeneses elicited by E. coli and S. aureus. Therefore, we studied, at several time points, the accumulation of neutrophils and the presence of the chemoattractant complement fragment C5a and of the cytokines interleukin-1β (IL-1β), tumor necrosis factor alpha, and IL-8 in milk after inoculation of E. colior S. aureus in lactating bovine udders. The low levels of C5a and the absence of cytokines in milk from S. aureus-infected cows, compared to the high levels found in milk from E. coli-infected animals, mirror the differences in the severities of the two inflammatory reactions. The cytokine deficit in milk after S. aureus inoculation in the lactating bovine mammary gland could contribute to the establishment of chronic mastitis. This result could help in the design of preventive or curative strategies against chronic mastitis.

Analysis of the Genetic Variability of Genes Encoding the RNA III-Activating Components Agr and TRAP in a Population of Staphylococcus aureus Strains Isolated from Cows with Mastitis

ABSTRACT The expression of Staphylococcus aureus virulence proteins is under the control of RNA III, a central pleiotropic regulator transcribed from the agr locus. RNA III is activated by at least two two-component systems, one encoded by the agr locus (AgrC-AgrA) and another encoded outside of this locus (TRAP-RAP). In this work, we developed new typing methods based on genes encoding these two systems, which we used to characterize a nonclonal population of S. aureus bovine mastitis isolates. Twelve agr restriction types were identified in this population, but the majority of strains (56.3%) were grouped in the R III-A1 type. No strain isolated from humans, whose agr sequence is available from GenBank, was found to belong to this major type. Restriction maps constructed for all of those agr variants allowed the linking of all types in an evolution scheme and their grouping in one of the four agr interference groups. This analysis indicates that groups 2, 3, and 4 probably evolved from the more frequently encountered type, which belongs to group 1. agr group 1 was also found to be the most prevalent (69.0% of the strains) and the most polymorphic interference group. By developing an agr group-specific multiplex PCR, we confirmed the above classification of strains in the agr interference groups. Four allelic variants of trap were also identified, indicating that this two-component system is also polymorphic. The majority of strains was grouped in the trap 1 type (71.8%). Whereas no relationships between agr group and trap types were found, strains of similar agr restriction type were also of similar trap type (with the exception of strains belonging to the agr R IV-A5 and R VI-A8 types). Our analysis indicates that S. aureus isolated from cows has predominantly a clonal structure and that the highly prevalent agr R III-A1, trap 1 type (56.3% of the strains) probably possesses a genetic background which endows it with superior ability to infect the bovine mammary gland.

Cells and Cytokines in Inflammatory Secretions of Bovine Mammary Gland

In response to invading bacteria, the mammary gland is protected by a variety of defence mechanisms, which can be separated into two distinct categories: innate immunity and specific immunity. Milk somatic cells consist of several cell types, including neutrophils, macrophages, lymphocytes and a smaller percentage of epithelial cells. In the healthy lactating mammary gland, macrophages are the predominant cell type whereas neutrophils are the major cell population during early inflammation. Following a bacteria invasion, neutrophil recruitment is elicited by inflammatory mediators that are produced in the infected gland by cells, possibly macrophages, activated by bacteria phagocytosis or responding to bacterial toxins or metabolites. Several cytokines, including interleukin- (IL-) 1 beta, IL-6, IL-8, tumour necrosis factor- (TNF-) alpha and interferon- (IFN-) gamma are known to be important to elicit the acute phase response and allow the accumulation of leukocytes at the site of infection. In addition to their role in early non-specific defences, macrophages also play a key role in the specific immune system, as antigen processing and presenting cells for the T cells. Few lymphocytes are found in milk of healthy glands where the predominant phenotype is CD8+ T cells. During the inflammatory reaction, T cells are recruited in milk and CD4+ cells become the predominant phenotype. The understanding of the specific and nonspecific immune mechanisms involved in the mammary gland defence against invading bacteria may lead to the development of new vaccines and to the use of cytokines to design immunomodulatory strategies for the control of bovine mastitis.

Leucotoxic Activities of Staphylococcus aureus Strains Isolated from Cows, Ewes, and Goats with Mastitis: Importance of LukM/LukF′-PV Leukotoxin

ABSTRACT 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.

Udder infection of goats by coagulase-negative staphylococci

Infection of udder halves by coagulase-negative staphylococci in seven commercial goat herds was studied in conjunction with the California Mastitis Test (CMT). Nine different species were identified and only 10% of strains belonged to groups which could not be identified with any of the known Staphylococcus species. The most prevalent species were Staphylococcus epidermidis (47.7%) and Staphylococcus caprae (19.7%). About half of the coagulase-negative staphylococcal infections gave negative CMT scores. The score was independent of the species of staphylococci involved and the stage of lactation. About 60% of the coagulase-negative staphylococcal species isolated were reisolated in the identical half udder during the following lactation.

Kinetics of cells and cytokines during immune-mediated inflammation in the mammary gland of cows systemically immunized with Staphylococcus aureus α-toxin

Abstract:Objective: To examine changes in inflammatory mediators, lymphocyte subpopulations and neutrophil activation that occur during an immune-mediated recruitment of neutrophils in the mammary gland.¶Subjects: 11 clinically healthy cows.¶Treatment: 5 cows received 2 subcutaneous injections of 30 μg of α-toxin of Staphylococcus aureus, two months apart. Three months after the last immunization, 5 μg of α-toxin were injected, via the teat end, in one randomly selected quarter of the 5 immunized cows and of the 6 unimmunized cows (control group).¶Methods: Blood and milk samples were collected at several times during 4 days post-challenge. Blood and milk cells were purified to be stained with specific mAbs and analysed by flow cytometry, or to be used for cytokine RT-PCR. Bovine serum albumin, haptoglobin, cytokines and C5a were also analysed in milk or plasma samples using radial immunodiffusion assay or ELISA.¶Results: Large amounts of cells (> 1 million/ml of milk) were recruited in the quarters of the immunized cows, whereas no recruitment occurred in the control group. In blood of immunized animals, haptoglobin was present and expression of surface adhesion molecules on neutrophils was modified whereas no change was observed concerning the lymphocyte subpopulations. On milk-derived neutrophils, the expression of CD11b and CD18 was upregulated compared to blood, in contrast to CD62L that was downregulated. The CD8+ cells were recruited as soon as 12 h post-challenge, in contrast to the CD4+ cells, 96 h post-challenge. No IL-1β, TNF-α, IL-8 and C5a were detected using ELISA. mRNA of IL-1α, IL-1β, IL-6, TNF-α, IL-8 and IL-12 were found in almost all the samples.¶Conclusions: The immunization triggered an early and massive neutrophil recruitment from the blood into the milk compartment as well as the recruitment of a cytotoxic/suppressor lymphocyte population during the early acute phase response. These results could help to devise new vaccinal strategies to fight against staphylococcal mastitis.¶

Immunogenicity in cows of Staphylococcus aureus type 5 capsular polysaccharide—ovalbumin conjugate

Six dairy cows were immunized subcutaneously with purified type 5 capsular polysaccharide (CP5) of Staphylococcus aureus or CP5-ovalbumin conjugate in Freunds incomplete adjuvant. The CP5 antibodies elicited were measured in sera and analysed with regard to isotypes by enzyme-linked immunosorbent assay. At the doses tested, the purified CP5 did not induce a humoral response in the cows. Immunization of two cows with the CP5-ovalbumin conjugate elicited a CP5 antibody response mainly of the IgG2 isotype, which culminated 4 weeks later. A second injection of conjugate, 3 months after the first one, resulted in a rapid and durable anti-CP5 response without exceeding the first antibody peak value. Intramammary infusion of purified CP5 failed to provoke an inflammatory response in the milk of the immunized cows. In contrast, a marked recruitment of cells was recorded in the milk of the sensitized cows after intramammary infusion of ovalbumin. These results demonstrate that injection of CP5-protein carrier conjugate in cows entails both antibody response against CP5 and carrier-specific recruitment of cells in milk of immunized animals.

Milk complement and the opsonophagocytosis and killing of Staphylococcus aureus mastitis isolates by bovine neutrophils

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 contribution of mammary infections by coagulase-negative staphylococci to the herd bulk milk somatic cell count.

Quarter foremilk samples were taken at 2–3 weekly intervals for several years in a 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.

International collaborative evaluation of the ATB 32 staph gallery for identification of the Staphylococcus species.

This international collaborative study evaluates a new system (ATB 32 Staph) for the identification of staphylococci taking into account the new novobiocin-sensitive and -resistant species reported. This study involved eight laboratories and 792 strains were tested. The reproducibility obtained for the cumulative results of the inter- and intra-laboratory tests was more than 90%. For 713 strains relevant of a species 95.5% were correctly identified by the system. Eight strains (1.2%) were misidentified and 24 strains (3.3%) were not identified. For 79 strains initially considered as not-classified, 62% were identified at the species level by the new system. The newer ATB 32 Staph gallery is a performant and useful method for routine identification of the currently described staphylococci species from clinical and animal origin.