Antibacterial activity and immunomodulatory effects on a bovine mammary epithelial cell line exerted by nisin A-producing Lactococcus lactis strains.

Twenty-nine strains of mastitis pathogens were used to study the antibacterial activity of the cell-free supernatants (CFS) of 25 strains of Lactococcus lactis ssp. lactis. Out of the tested strains, only the CFS of L. lactis LL11 and SL153 were active, inhibiting and killing most of the pathogens. By means of ultra-performance liquid chromatography/high resolution mass spectrometry, they were shown to produce nisin A, a class I bacteriocin. A variable sensitivity to nisin A-containing CFS was observed among Streptococcus uberis and Enterococcus faecalis strains. Nonetheless, Streptococcus agalactiae, Strep. uberis, and E. faecalis displayed high minimum inhibitory concentration values, reaching 384 arbitrary units/mL. Interestingly, the minimum inhibitory values and the bactericidal concentrations were almost identical among them for each of the 2 stains, LL11 and SL153. Staphylococci were, on average, less sensitive than streptococci, but the 2 CFS inhibited and killed, at different dilutions, strains of methicillin-resistant Staphylococcus aureus. The immune response to nisin A-containing CFS was tested using the bovine mammary epithelial cell line BME-UV1. Application of CFS did not damage epithelial integrity, as demonstrated by the higher activity of N-acetyl-ß-d-glucosaminidase (NAGase) and lysozyme inside the cells, in both treated and control samples. On the other hand, the increase of released NAGase after 15 to 24h of treatment with LL11 or SL153 live cultures demonstrated an inflammatory response of epithelial cells. Similarly, a significantly higher lysozyme activity was detected in the cells treated with LL11 live culture confirming the stimulation of lysosomal activity. The treatment of epithelial cells with SL153 live culture induced a significant tumor necrosis factor-α downregulation in the cells, but did not influence IL-8 expression. The control of tumor necrosis factor-α release could be an interesting approach to reduce the symptoms linked to clinical intramammary infections. Due to their antibacterial activity and to the stimulation of lysosomal activity of mammary epithelial cells, the L. lactis strains SL153 and LL11 could be of interest for the development of alternative intramammary treatments to control cow mastitis.

Evaluation of biofilm formation using milk in a flow cell model and microarray characterization of Staphylococcus aureus strains from bovine mastitis.

It was hypothesized that biofilm could play an important role in the establishment of chronic Staphylococcus aureus bovine mastitis. The in vitro evaluation of biofilm formation can be performed either in closed/static or in flow-based systems. Efforts have been made to characterize the biofilm-forming ability of S. aureus mastitis isolates, however most authors used static systems and matrices other than UHT milk. It is not clear whether such results could be extrapolated to the mammary gland environment. Therefore, the present study aimed to investigate the biofilm-forming ability of S. aureus strains from subclinical bovine mastitis using the static method and a flow-based one. One hundred and twelve strains were tested by the classic tissue culture plate assay (TCP) and 30 out of them were also tested by a dynamic semi-quantitative assay using commercial UHT milk as culture medium (Milk Flow Culture, MFC) or Tryptic Soy Broth as control medium (TS Flow Culture, TSFC). Only 6 (20%) strains formed biofilm in milk under flow conditions, while 36.6% were considered biofilm-producers in TCP, and 93.3% produced biofilm in TSFC. No agreement was found between TCP, MFC and TSFC results. The association between strain genetic profile, determined by microarray, and biofilm-forming ability in milk was evaluated. Biofilm formation in MFC was significantly associated with the presence of those genes commonly found in bovine-associated strains, assigned to clonal complexes typically detected in mastitis. Based on our results, biofilm-forming potential of bovine strains should be critically analysed and tested applying conditions similar to mammary environment.

Differential cell count as an alternative method to diagnose dairy cow mastitis.

Changes in relative cell proportions occurring in diseased mammary glands of dairy cows can be determined using differential cell count (DCC). The present study was carried out in 2 consecutive trials, with 2 goals: (a) to test the consistency of DCC results on subsequent days, and (b) to establish an effective cutoff value for the diagnosis of mastitis. In the first trial, quarter milk and blood samples were taken from 8 healthy cows for 5 consecutive days. Milk samples were tested by somatic cell count (SCC) and bacteriological analysis, and DCC was performed on blood and milk samples by flow cytometer. In the second trial, 16 animals were randomly selected from a different herd and quarter milk samples taken on 3 consecutive milkings. All samples were cyto-bacteriologically analyzed and DCC was performed on the second sampling. In the first trial, mean SCC was 77,770 cells/mL and 4 samples were bacteriologically positive. No fixed or random effect had a significant influence on percentages of individual cell populations or ratios in blood or milk. A cutoff value of 0.495 for logarithmic polymorphonuclear neutrophilic leukocyte:lymphocyte ratio was established. Mean SCC of milk samples collected in the second trial was 543,230 cells/mL, and infection was detected in 53.1% of quarters, mostly caused by Staphylococcus aureus. When the cutoff value was applied to the data along with SCC, sensitivity and specificity of the diagnostic method were 97.3 and 92.3%, respectively.