Short Communication: Effects of dietary Bacillus subtilis PB6 supplementation to mid-lactation dairy cows on local inflammatory responses during an intramammary lipopolysaccharide challenge

Objectives of this study were to evaluate the effects of dietary Bacillus subtilis PB6 supplementation on inflammatory mediators in milk during an intramammary lipopolysaccharide (LPS) challenge in dairy cows.A secondary objective was to determine if repeated LPS challenges induced the development of innate immune memory and immunological tolerance.Mid-lactation Holstein cows (n = 20; 147 48 DIM; 2.2 0.93 parity; 42.1 7.6 kg/d of milk; mean SD) were enrolled in a crossover design.Treatments included Bacillus subtilis PB6 (BSP) or limestone (CON) at 13 g/d, which were top-dressed daily onto a basal lactating TMR for 28-d periods.An intramammary LPS challenge (10 g, Escherichia coli O111:B4) was conducted in a rear quarter on d 24 of each period.Milk samples were collected at 0, 3, 12, and 24 h relative to the intramammary LPS challenge from the challenged quarter to measure SCC and cytokines.For cytokines, milk samples were treated with rennet and centrifuged to obtain whey, and 13 cytokines/chemokines were measured using a multiplex assay (IFN, IL-1, IL-1, IL-4, IL-6, IL-10, IL-17A, IL-36RA, IP-10, MCP-1, MIP-1, TNF-, and VEGF-A).Linear mixed models included the fixed effects of treatment, period, and time, and their interactions, and the random effects of cow nested within period, block, and block by period.Dietary BSP supplementation increased SCS at 3 h but decreased SCS at 12 h during period 1 as compared with the CON; there were no treatment effects on SCS during period 2. Dietary BSP increased whey MCP-1 (monocyte chemokine) at 3, 12, and 24 h relative to the LPS challenge during period 1; however, during period 2, dietary BSP decreased whey MCP-1 at 3 and 12 h as compared with CON.These data suggest that dietary BSP supplementation may have enhanced innate immune training during period 1 and these effects carried over into the CON cows during period 2. Independent of treatment sequence, period 1 had greater concentrations of whey IL-1 and IL-1 at 3 h following the LPS challenge as compared with period 2, suggesting the development of endotoxin tolerance.In conclusion, our data suggests that dietary BSP supplementation may alter whey MCP-1 concentrations, which may have influenced immune cell trafficking to the mammary gland during an intramammary LPS challenge.Period effects point to the development of endotoxin tolerance in cows receiving 2 intramammary LPS challenges.