Immune cell counts and signaling in body fluids of cows vaccinated against Clostridium difficile.

BACKGROUND: New treatment options are needed to prevent relapses following failed antibiotic therapies of Clostridium difficile infections (CDI) in humans. The concomitant therapy with an anti-C. difficile IgA containing whey protein concentrate can support the sustainable recovery of CDI patients. For 31 weeks, nine dairy cows were continuously vaccinated with several anti-C. difficile vaccines by certain routes of administration to produce anti-C. difficile IgA enriched milk. The study aimed at finding decisive differences between low responder (LR) and high responder (HR) cows (> 8.0 µg ml-1 total milk C. difficile specific IgA) concerning their immune response to vaccination on cellular and molecular biological levels. RESULTS: The results of total and differential cell counting (DCC) in blood and milk and the outcomes of the gene expression analysis of selected immune factors were assessed relating to the usage of two vaccine batches for injection (MucoCD-I batch A and B), marking two immunization (IM) periods, and compared to a control group (Ctr). The MucoCD-I batch A caused short-term leukopenia followed by leukocytosis in the blood of LR and HR. The total somatic cell counts in milk were not altered by the treatment. The DCC revealed that the leukocytes of the treated groups were partly impaired by the treatment. The gene expression analysis exposed cumulative and sustainable differences (p < 0.05) between LR and HR for the genes encoding for lactoferrin, CXCL8, IL1ß, IL2, IL6, IL12ß, IFNγ, CD4 and CD163. The regulation of the epithelial IgA cell receptor PIGR was not impaired by the IM. In contrast to the vaccination with MucoCD-I batch A, the second IM period with MucoCD-I batch B resulted in mitigation and synchronization of the treated groups' immune responses. CONCLUSIONS: The inversely regulated cytokines in the blood and milk cells of the treated groups led to a variously directed, local T cell response resulting in their different production intensities of C. difficile specific IgA in milk.

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.