Short communication: Differential loss of bovine mammary epithelial barrier integrity in response to lipopolysaccharide and lipoteichoic acid.

In the mammary gland, the blood-milk barrier prevents an uncontrolled intermixture of blood and milk constituents and hence maintains the osmotic gradient to draw water into the mammary secretion. During mastitis, the permeability of the blood-milk barrier is increased, which is reflected by the transfer of blood constituents into milk and vice versa. In this study, we aimed to investigate changes in the barrier function of mammary epithelial cells in vitro as induced by cell wall components of different pathogens. Primary bovine mammary epithelial cells from 3 different cows were grown separately on Transwell (Corning Inc., Corning, NY) inserts. The formation of tight junctions between adjacent epithelial cells was shown by transmission electron microscopy and by immunofluorescence staining of the tight junction protein zona occludens-1. The integrity of the epithelial barrier was assayed by means of transepithelial electrical resistance, as well as by diffusion of the fluorophore Lucifer yellow across the cell layer. The release of lactate dehydrogenase (LDH) was used as an indicator for cytotoxic effects. In response to a 24-h challenge with bacterial endotoxin, barrier integrity was reduced after 3 or 7h, respectively, in response to 0.5mg/mL lipopolysaccharide (LPS) from Escherichia coli or 20mg/mL lipoteichoic acid (LTA) from Staphylococcus aureus. No paracellular leakage was observed in response to 0.2mg/mL LPS or 2mg/mL LTA. Although LPS and LTA affected barrier permeability, most likely by opening the tight junctions, only LPS caused cell damage, reflected by increased LDH concentrations in cell culture medium. These results prove a pathogen-specific loss of blood-milk barrier integrity during mastitis, which is characterized by tight junction opening by both LPS and LTA and by additional epithelial cell destruction through LPS.

The immune response of bovine mammary epithelial cells to live or heat-inactivated Mycoplasma bovis.

Mycoplasma bovis is an emerging bacterial agent causing bovine mastitis. Although these cell wall-free bacteria lack classical virulence factors, they are able to activate the immune system of the host. However, effects on the bovine mammary immune system are not yet well characterized and detailed knowledge would improve the prevention and therapy of mycoplasmal mastitis. The aim of this study was to investigate the immunogenic effects of M. bovis on the mammary gland in an established primary bovine mammary epithelial cell (bMEC) culture system. Primary bMEC of four different cows were challenged with live and heat-inactivated M. bovis strain JF4278 isolated from acute bovine mastitis, as well as with the type strain PG45. The immune response was evaluated 6 and 24h after mycoplasmal challenge by measuring the relative mRNA expression of selected immune factors by quantitative PCR. M. bovis triggered an immune response in bMEC, reflected by the upregulation of tumor necrosis factor-α, interleukin(IL)-1ß, IL-6, IL-8, lactoferrin, Toll-like receptor-2, RANTES, and serum amyloid A mRNA. Interestingly, this cellular reaction was only observed in response to live, but not to heat-inactivated M. bovis, in contrast to other bacterial pathogens of mastitis such as Staphylococcus aureus. This study provides evidence that bMEC exhibit a strong inflammatory reaction in response to live M. bovis. The lack of a cellular response to heat-inactivated M. bovis supports the current hypothesis that mycoplasmas activate the immune system through secreted secondary metabolites.

Different chronological patterns of appearance of blood derived milk components during mastitis indicate different mechanisms of transfer from blood into milk.

This study aimed to describe chronological patterns of changes of various candidate blood components in milk during the acute phase of a mammary immune response in detail. Eight dairy cows were challenged with Escherichia coli lipopolysaccharide in one udder quarter. Milk from challenged and control quarters and blood samples were taken before, and 1 and 2 h after challenge and then every 15 min until 5 h after challenge. The SCC, serum albumin, immunoglobulin (Ig)G1, IgG2, lactate dehydrogenase (LDH), and L-lactate in milk and blood, and α-lactalbumin in blood were analysed. All selected parameters in milk increased in challenged quarters but did not increase in control quarters. Milk IgG1, IgG2, serum albumin, and LDH were already significantly increased at 2 h after challenge whereas a significant increase of SCC was detectable at 2.75 h and L-lactate was increased at 2.25 h after challenge. In blood L-lactate was increased at 3.75 h after challenge, however, other factors in blood did not change significantly within the 5 h of experiment. In conclusion, the increase of blood components in milk during inflammation follows two different patterns: There is a rapid increase for IgG1, IgG2, or LDH, before the increase of SCC, and their concentrations reach a plateau within 3 h. On the other hand, SCC and L-lactate show a slower but consistent increase not reaching a plateau within 5 h after LPS challenge. L-lactate increases to higher concentrations in milk than in blood. This clearly shows that the increase of blood components follows different patterns and is therefore a controlled and compound-specific process and not exclusively an unspecific type of leakage.

The inflammatory response of primary bovine mammary epithelial cells to Staphylococcus aureus strains is linked to the bacterial phenotype.

Staphylococcus aureus is a major mastitis-causing pathogen in dairy cows. The latex agglutination-based Staphaurex test allows bovine S. aureus strains to be grouped into Staphaurex latex agglutination test (SLAT)-negative [SLAT(-)] and SLAT-positive [SLAT(+)] isolates. Virulence and resistance gene profiles within SLAT(-) isolates are highly similar, but differ largely from those of SLAT(+) isolates. Notably, specific genetic changes in important virulence factors were detected in SLAT(-) isolates. Based on the molecular data, it is assumed that SLAT(+) strains are more virulent than SLAT(-) strains. The objective of this study was to investigate if SLAT(-) and SLAT(+) strains can differentially induce an immune response with regard to their adhesive capacity to epithelial cells in the mammary gland and in turn, could play a role in the course of mastitis. Primary bovine mammary epithelial cells (bMEC) were challenged with suspensions of heat inactivated SLAT(+) (n = 3) and SLAT(-) (n = 3) strains isolated from clinical bovine mastitis cases. After 1, 6, and 24 h, cells were harvested and mRNA expression of inflammatory mediators (TNF-α, IL-1ß, IL-8, RANTES, SAA, lactoferrin, GM-CSF, COX-2, and TLR-2) was evaluated by reverse transcription and quantitative PCR. Transcription (ΔΔCT) of most measured factors was induced in challenged bMEC for 6 and 24 h. Interestingly, relative mRNA levels were higher (P<0.05) in response to SLAT(+) compared to SLAT(-) strains. In addition, adhesion assays on bMEC also showed significant differences between SLAT(+) and SLAT(-) strains. The present study clearly shows that these two S. aureus strain types cause a differential immune response of bMEC and exhibit differences in their adhesion capacity in vitro. This could reflect differences in the severity of mastitis that the different strain types may induce.