Stimulated enrichment of Clostridium difficile specific IgA in mature cow's milk.

Cow milk products enriched with Clostridium difficile (C. diff.) specific IgA are possible alternative therapeutics against C. diff. associated diarrhea. A persistently high level of C. diff. specific IgA in mature milk triggered by continuous immunizations of dairy cows against C. diff. was hypothesized. Nine Brown Swiss cows were repeatedly vaccinated against C. diff. and divided into low responder (LR) and high responder (HR) cows, as measured by their production of anti-C. diff. specific IgA in milk (threshold: 8.0 µg anti-C. diff. specific IgA/mL on average). Total and C. diff. specific IgA were quantified in bovine milk and blood using a sandwich ELISA. Important milk production factors were analyzed per lactation stage. Milk yield, milk fats and proteins were significantly different (P < 0.05) in the early lactation stage when the treated with the untreated cows (n = 30) were compared. In contrast to the "before treatment control" values, the HR's milk anti-C. diff. IgA was approximately 80% higher at any lactation stage, and the HR's total milk IgA increased up to 72% in the late lactation stage. The LR's total milk IgA differed from the baseline by roughly 47% only in the late lactation stage. The total and specific IgA contents in milk were more influenced by the anti-C. diff. immunizations than in blood. The correlations between anti-C. diff. specific IgA, total IgA and the main production factors in milk were classified as weak (I r I < 0.5), except for the close relation of anti-C. diff. specific IgA and total IgA (r = 0.69). To conclude, a sustainable C. diff. specific IgA enrichment in milk can be achieved by continuous immunization of dairy cows, provided a potent and well-formulated anti-C. diff. vaccine is given to dairy cows preselected due to their proven anti-C. diff. receptivity.

Establishment of a 3D cell culture model of primary bovine mammary epithelial cells extracted from fresh milk.

For the investigation of molecular processes underlying diseases of the bovine mammary gland, primary bovine mammary epithelial cells (pbMEC) are used. They are known to contribute to the innate immune system of the bovine mammary gland. The functionality of pbMEC depends on the maintenance of in vivo characteristics. So far, the optimization of pbMEC culture conditions was intended in a variety of experiments. For this purpose, most of the studies used stable cell lines or primary cells obtained from udder biopsies of slaughtered animals. By contrast, within our study, pbMEC of healthy and first lactating Brown Swiss cows were non-invasively isolated from fresh milk. The non-invasively isolated pbMEC were cultivated on the extracellular matrix-like scaffold Matrigel®. Further, they were challenged with different compositions of proliferation media, containing lactogenic hormones and/or the essential amino acid L-lysine. Changes in expression levels of genes coding for milk proteins and for components of the janus kinase/signal transducers and activators of transcription (JAK-STAT) and mTOR pathways were analyzed by RT-qPCR. The secreted proteins were analyzed by LC-MS/MS measurements. We showed for the first time the establishment of a physiologically functional 3D cell culture model of pbMEC isolated from fresh milk. This represents a primary cell culture model system, based on non-invasive cell collection, that can be used to unravel physiological processes in an unbiased manner.

Effect of the Ketone Body Beta-Hydroxybutyrate on the Innate Defense Capability of Primary Bovine Mammary Epithelial Cells.

Negative energy balance and ketosis are thought to cause impaired immune function and to increase the risk of clinical mastitis in dairy cows. The present in vitro study aimed to investigate the effect of elevated levels of the predominant ketone body ß-hydroxybutyrate on the innate defense capability of primary bovine mammary epithelial cells (pbMEC) challenged with the mastitis pathogen Escherichia coli (E. coli). Therefore, pbMEC of healthy dairy cows in mid- lactation were isolated from milk and challenged in culture with 3 mM BHBA and E. coli. pbMEC stimulated with E. coli for 6 h or 30 h showed an up-regulation of several innate immune genes, whereas co-stimulation of pbMEC with 3 mM BHBA and E. coli resulted in the down-regulation of CCL2, SAA3, LF and C3 gene expression compared to the challenge with solely the bacterial stimulus. These results indicated that increased BHBA concentrations may be partially responsible for the higher mastitis susceptibility of dairy cows in early lactation. Elevated levels of BHBA in blood and milk during negative energy balance and ketosis are likely to impair innate immune function in the bovine mammary gland by attenuating the expression of a broad range of innate immune genes.