María S. Renna

Detection of Staphylococcus aureus adhesion and biofilm-producing genes and their expression during internalization in bovine mammary epithelial cells.

Staphylococcus aureus is one of the most prevalent pathogens isolated from bovine mastitis, causing chronic intramammary infections (IMI) that limit profitable dairying. The course of infection is often associated with factors both related to the host and the bacterium. Aims of this study were to select S. aureus isolates from bovine IMI with different genotypic profiles harboring genes involved in adherence and biofilm production, to determine the behavior of these strains in contact with bovine mammary epithelial cells (MAC-T) and the expression of those genes during bacterial-cell early interactions. The genetic diversity of 20 S. aureus strains that were isolated from milk samples taken from cows with persistent-P and non-persistent-NP IMI was high, discriminated into 13 fingerprint groups. The occurrence of genes coding for S. aureus surface proteins (clfA, clfB, fnbA, fnbB, fib, cna) and biofilm formation (icaA, icaD, icaC, bap) and in vitro biofilm-forming ability was not related to strain clinical origin (NP or P). Internalization of S. aureus into MAC-T cells was strain-dependent and internalized bacteria overexpressed adherence and biofilm-forming genes compared with those that remained in the supernatant of co-cultures; particularly those genes encoding FnBPs and IcaD. Strains yielding highest invasion percentages were those able to overexpress fnBP, irrespectively of the presence of other evaluated genes. Strains from NP IMI showed a greater multiplication capacity in vitro compared with strains from P IMI. These results provide new insights about S. aureus differential gene expression of adhesion-internalization factors during early interaction with mammary epithelial cells.

Staphylococcus aureus chronic intramammary infection modifies protein expression of transforming growth factor beta (TGF-β) subfamily components during active involution.

The objectives of this study were to determine whether Staphylococcus aureus chronic intramammary infection (IMI) influences protein expression of TGF-β subfamily components and collagen I and to examine the histomorphometric changes that occur in mammary stroma and parenchyma during active mammary gland involution. Twenty-one Holstein non-pregnant cows in late lactation either uninfected or with chronic natural S. aureus IMI were included in this study. Cows were slaughtered at 7, 14 and 21d after cessation of milking and samples for immunohistochemical and morphometric analysis were taken. Protein expression of TGF-β1, TGF-β2 and TGF-β3 was significantly higher in chronically infected quarters than in uninfected controls at the three involution stages studied. Immunostaining of TGF-βR1 and TGF-βR3 and collagen I was significantly higher in S. aureus-infected quarters than in uninfected controls at every involution time evaluated. The percentages of tissue area composed of parenchyma and intralobular stroma were significantly higher in S. aureus-infected than in uninfected quarters. Chronic S. aureus mastitis modifies protein expression of the three TGF-β isoforms and type 1 and 3 receptors, which was associated with changes directed to limit the scope of inflammation and injury to the host.

Intramammary infusion of Panax ginseng extract in the bovine mammary gland at cessation of milking modifies components of the insulin-like growth factor system during involution

The objective of this study was to evaluate the effects of a single intramammary infusion of Panax ginseng extract (GS) on insulin-like growth factors (IGF) in bovine mammary gland during early involution. Eight mammary quarters from six nonpregnant cows in late lactation were infused with 10 mL of ginseng extract solution (3 mg/mL), six quarters were treated with 10 mL of placebo (vehicle alone) and six quarters were maintained as uninoculated controls. Milking was interrupted after infusion. Concentrations of IGF1 in mammary secretions were higher in GS-treated quarters than in placebo and uninoculated control quarters at 24, 48 and 72 h post-treatment (p<0.05). Treatment with GS did not affect mammary secretion of IGF2 (p=0.942). At 7 d of post-lactational involution, a decrease of immunostained area and mRNA expression for IGF1 was observed in mammary tissue of GS-treated quarters compared with placebo-treated quarters and uninoculated controls (p<0.05). The IGF2 immunostained area and mRNA expression for this growth factor were not affected by GS treatment (p=0.216 and p=0.785, respectively). An increase in protein levels and mRNA expression in mammary tissue of IGFBP3, IGFBP4 and IGFBP5 was observed in GS-treated quarters compared with placebo-treated quarters and uninoculated controls (p<0.05). These results provide evidence that intramammary inoculation of GS extract at cessation of milking may promote early mammary involution through the inhibition of IGF1 local production and bioavailability.

Intramammary infusion of Panax ginseng extract in bovine mammary gland at cessation of milking induces changes in the expression of toll-like receptors, MyD88 and NF-kB during early involution

The purposes of this study were to explore TLR2 and TLR4 participation and MyD88 and NF-κB activation in bovine mammary glands (BMG) treated with Panax ginseng (PG) at involution and verify the effect of PG in the cytokine expression. Quarters were infused at the end of lactation with PG solution (3 mg/ml), placebo or kept as uninoculated controls. Cows were slaughtered at 7 d after cessation of milking and mammary tissue samples were taken. A significant increase of TLR2, TLR4, MyD88, NF-κB, IL-1β, IL-6 and TGF-β1 mRNA expression was observed in PG-treated quarters. Immunostaining of TLR2 and TLR4 was significantly higher in PG mammary tissues. The percentages of immunopositive cells for NF-κB-p65 were significantly higher in PG-treated quarters. The BMG responded to PG extract components possibly by TLR2 and TLR4 signaling pathway. These results provide an insight into potential mechanisms by which PG stimulates innate immunity during BMG involution.

Comparison of phenotypic tests for detecting penicillin G resistance with presence of blaZ gene in Staphylococcus aureus isolated from bovine intramammary infections.

Few studies have described the relationship between genotypic and phenotypic methods for detecting penicillin resistance in Staphylococcus aureus isolated from bovine intramammary infection (IMI). Six phenotypic methods for penicillinase detection were compared with a genotypic method testing the presence of the β-lactamase gene blaZ in Staph. aureus (n = 150) isolated from bovine IMI. Highest sensitivities and specificities were observed for disk diffusion (DD) (93 and 97.4%), minimum inhibitory concentration (MIC) (90.3 and 97.4%), Cefinase™ (85.9 and 97.4%) and Diatabs™ (85.7 and 98.7%). The estimated cut-off points estimated in the present study can be considered close to the ones indicated by CLSI (2013). The molecular detection of blaZ gene is the only method that may indicate the real or potential capacity of producing β-lactamase in Staph. aureus. Considering that from a clinical standpoint a false negative result from a phenotypic test is the most unfavourable situation, a combination of standard DD with Diatabs™ or Cefinase™ should be performed by routine mastitis laboratories to minimise false negative results.

Functional role of antibodies generated in heifers through immunization with Staphylococcus aureus vaccines in invasion and phagocytosis assays

A successful Staphylococcus aureus vaccine should elicit a long-term antibody response that prevents establishment of the infection. The aim of the present study was to evaluate the functional role of antibodies raised against different S. aureus CP5 vaccines in invasion to bovine mammary epithelial cells (MAC-T) and phagocytosis by bovine milk macrophages in vitro. Sera and whey from cows immunized with a whole-cell S. aureus CP5 vaccine adjuvanted with Al(OH)3 or with ISCOM Matrix, significantly reduced internalization of S. aureus in MAC-T cells without significant differences between both groups. The effect of antibodies generated by a S. aureus whole-cell and a lysate vaccine formulated with ISCOM Matrix was also evaluated. Sera and whey from both immunized groups significantly reduced S. aureus internalization in MAC-T cells without significant differences between both groups. Whey antibodies against whole-cell and lysate vaccines were also able to inhibit internalization in MAC-T cells of a heterologous S. aureus strain. In addition, sera from animals vaccinated with S. aureus lysate or bacterin promoted milk macrophage phagocytosis. These results provide an insight into the potential mechanisms by which these vaccines can afford protection to the mammary gland against S. aureus intramammary infection.

Intramammary inoculation of Panax ginseng plays an immunoprotective role in Staphylococcus aureus infection in a murine model.

The immunoprotective effect of Panax ginseng (Pg) extract was investigated in a mouse mastitis model. Lactating female mice were intramammarily inoculated with Pg or placebo, and then were challenged with S. aureus, while other group was inoculated with S. aureus alone. The number of bacteria recovered from mammary glands was significantly lower in Pg-treated S. aureus-infected mice (group I) compared with placebo-treated S. aureus-infected mice (group II) and S. aureus-infected mice (group III). The mRNA expression of TLR2, TLR4, IL-1α and TNF-α was influenced by treatment; being the transcript levels for all genes higher in group I compared with group II and III. Activation of NF-κB and the number of monocytes-macrophages in mammary gland tissue was significantly increased in group I compared with group II and III. Pg extract was able to trigger an adequate immune response to confront an infection demonstrating its protective effect and potential for preventing bovine intramammary infections.

Immune response of Staphylococcus aureus strains in a mouse mastitis model is linked to adaptive capacity and genotypic profiles

Staphylococcus aureus is one of the most frequently isolated major pathogens from intramammary infections (IMI) worldwide. The mechanisms by which S. aureus IMI are established and maintained in dairy cows involve both bacterial escape strategies and modulation of the host immune response. Moreover, it was shown that different S. aureus strains have varying effects on the immune response. The aim of this study was to investigate the immune response in a mouse mastitis model of two S. aureus strains isolated from bovine IMI with different clinical manifestation (persistent-P or non-persistent-NP), phenotypic and genotypic profile. Both strains were capable of establishing an IMI after 264h post inoculation (pi). Strain A (NP) showed a more aggressive behaviour than strain B (P) at early stages of IMI, while strain B multiplied initially at a lower rate but increased its replication capacity from 120h pi to the end of the study (264h pi). Strain A triggered a stronger initial inflammatory response compared with strain B inducing higher gene and protein expression of TLR2, NF-κB activation and higher gene expression of IL-1α at initial stage of IMI (6-12h pi) but inducing extensive mammary tissue damage. Immune cells response was different for each S. aureus strain throughout the course of infection, showing mammary glands inoculated with strain A greater initial immune cells stimulation compared with strain B and then a second immune cells stimulation (from 120 to 264h pi) represented by monocytes-macrophages, T and B lymphocytes, mainly stimulated by strain B, consistent with inflammatory process becoming chronic. Strain-specific pathogenicity observed underscores the importance of pathogen factors in the progression of the infectious process. These results contribute to increase the available information on host-pathogen interaction and point out for the need of further research to expand the knowledge about these interactions for developing new strategies to intervene in the IMI progress.

Proliferation-apoptosis balance in Staphylococcus aureus chronically infected bovine mammary glands during involution

The objective of this study was to determine whether Staphylococcus aureus chronic intramammary infection (IMI) influences expression of proteins related to regulation of proliferation and apoptosis processes and proliferation/apoptosis index during active involution in bovine mammary gland. Twenty-one Holstein non-pregnant cows in late lactation either uninfected or with chronic naturally acquired S. aureus IMI were included in this study. Cows were slaughtered at 7, 14 and 21 d after cessation of milking and samples for immunohistochemical analysis were taken. Protein expression of Bcl-2, Bax, Fas and active caspase-3 in mammary tissue was significantly affected by chronic S. aureus IMI, all showing increased immunoexpression in S. aureus-infected quarters at all involution stages. The percentage of apoptotic cells was increased by IMI in both mammary parenchyma and stroma, and the percentage of parenchymal and stromal cell proliferation was also increased. The proliferation/apoptosis ratio was significantly increased by IMI only in stromal cells. This imbalance to favour proliferation in S. aureus-infected mammary quarters could be one of the underlying causes that induce aberrant involution with permanence of nonsecretory tissue and increase of stromal components.

Panax ginseng extract reduces Staphylococcus aureus internalization into bovine mammary epithelial cells but does not affect macrophages phagocytic activity

Panax ginseng extract (PGe) has been shown to possess immunomodulatory effects in healthy dairy cows at drying off and to trigger an adequate immune response to protect from an experimental intramammary infection (IMI) with Staphylococcus aureus in a murine model. S. aureus is one of the major pathogens isolated from bovine IMI; being capable to invade and survive within mammary epithelial cells. However, the precise mechanism by which PGe interacts with bovine mammary epithelial cells (MAC-T) and bovine macrophages in the course of a S. aureus infection remains unclear. We evaluated the effect of PGe on MAC-T cytokine response and on the internalization of S. aureus into MAC-T. In addition, we evaluated the effect of PGe on the phagocytic activity of macrophages isolated from bovine mammary secretions. Results shown that MAC-T cells TLR4 and NF-κB mRNA expression was not affected by PGe at all evaluated times. IL-6 mRNA expression and protein level and IL-4 protein level were significantly induced in MAC-T treated with 3 mg/ml of PGe. PGe at 3 mg/ml reduced significantly the internalization of two S. aureus strains in MAC-T. In addition, PGe did not affect the percentage of phagocytosis and the NO and ROS production of macrophages co-cultured with two strains of S. aureus. These results, obtained in in vitro models together with those obtained in in vivo previous studies carried out in bovines and mice can contribute to improve the understanding of the effects of PGe following inoculation in bovine mammary glands.