Treatment scheme of the pbMEC <i>in vitro</i>.

The green arrow indicates the untreated control samples that were taken at every relevant time-point (0 h, 24 h, 30 h and 54 h). The blue arrow indicates the BHBA treatment time-points. The pbMEC that were only treated with 3 mM BHBA were also sampled at every relevant time-point (24 h, 30 h and 54 h). The red arrows indicate the two treatment intervals for the E. coli treatment (6 h and 30 h). It can be clearly seen that within the co-stimulated pbMEC, a 24 h adaptation phase to 3 mM BHBA preceded the bacterial treatment, so that pbMEC stimulated with E. coli for 6 h, obtained a total BHBA treatment period for 30 h and pbMEC stimulated with E. coli for 30 h obtained a total BHBA treatment period of 54 h.</p

Comparison of the RT-qPCR data of LF and CCL2 with the LF and CCL2 content in pbMEC cell culture supernatants.

<p>The fold changes of the LF gene expression (A) indicated a significant down-regulation of LF gene expression when pbMEC where co-stimulated with <i>E</i>. <i>coli</i> and 3 mM BHBA. The same effect could be detected within the competitive LF ELISA (B) of pbMEC cell culture supernatants. The amount of secreted LF decreased significantly in case of the co-stimulation. The distinct and significant down-regulation in CCL2 gene expression (C) could also be confirmed by the results of the bovine CCL2 VetSet^™ ELISA Kit (D). The CCL2 gene expression and the amount of secreted protein decreased distinctly as well as significantly within the co-stimulatory group. Significant changes: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, distinct changes: + 0.1 ≤ p ≤ 0.05.</p

Primer for RT-qPCR measurements.

<p>Primer for RT-qPCR measurements.</p

Attenuation of the gene expression through the co-stimulation of pbMEC with <i>E</i>. <i>coli</i> and 3 mM BHBA.

Fold change of (A) the chemokine CCL2, (B) the milk protein CSN3, (C) the complement component C3, (D) the antimicrobial peptide LF, (E 1–2) the acute phase protein SAA3, and (F) the inflammatory cytokine IL6. Significant changes in the gene expression between the E. coli treated pbMEC and the pbMEC co-stimulated with E. coli and 3 mM BHBA are indicated by stars: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.</p

IC of pbMEC.

<p>The typical cobblestone-like morphology and the epithelial character were confirmed, by the positive cytokeratin staining, the insert shows the negative control. [Leica light microscope, magnification x200].</p

Fold changes in gene expression upon immune stimulation.

<p>Fold changes in gene expression upon immune stimulation.</p

Immunization schedule.

<p>Immunization schedule.</p

Diseases of treated cows and the TW per vaccinated cow in which diseases emerged.

<p>Diseases of treated cows and the TW per vaccinated cow in which diseases emerged.</p

Anti-<i>C</i>. <i>diff</i>. milk IgA contents in the different lactation stages.

<p>BTC means the "before treatment control" value of all cows in frame for vaccination. The lactation stages are defined as “early” for ≤ 100 DiL, “mid” for 101–200 DiL and “late” for ≥ 201 DiL. On the ordinate, <i>C</i>. <i>diff</i>. specific milk IgA concentrations are depicted as LSM ± SD and presented comparatively for the BTC (n = 9, black bar), low responder (LR, n = 4, cross-striped bars) and high responder (HR, n = 5, dotted bars) groups. Substantial or highly significant differences versus BTC and between LR and HR in the same lactation stage are marked by brackets with one (P < 0.05) or two asterisks (P < 0.01). Significant differences within the same group related to the early lactation stage are shown in lower case letters (a, b) for P < 0.05.</p

Anti-<i>C</i>. <i>diff</i>. IgA concentrations in milk and in blood during the 31-week treatment period.

<p>The ordinate values in milk (solid lines, top) and in blood (broken lines, bottom) are shown as LSM ± SD per treated groups, the low responder (LR, n = 4, triangles) and the high responder (HR, n = 5, circles). Substantial or highly significant differences between both groups at the same point in time are marked by one (P < 0.05) or two asterisks (P < 0.01). The gray bars indicate the one-time application of <i>MucoCD-I</i> vaccine batch A in TW 3 and the first use of vaccine batch B for injection in TW 17. The vaccination routes were nasal (N), percutaneous (PC), subcutaneous (SC) and intracutaneous (IC). The injected <i>MucoCD-I</i> vaccine was uniquely supplemented with incomplete Freund’s adjuvant (iFA).</p