Viktoria Neubauer Viktoria Neubauer Siska Aditya Siska Aditya Narciso M. Quijada Narciso M. Quijada Stefanie Urimare Wetzels Stefanie Urimare Wetzels Monika Dzieciol Monika Dzieciol Poulad Pourazad Poulad Pourazad Qendrim Zebeli Qendrim Zebeli Evelyne Selberherr Evelyne Selberherr Springer 2026 Background Lipopolysaccharides (LPS) from pathogenic Gram-negative bacteria play a key role in the pathophysiology of mastitis. Subacute ruminal acidosis (SARA) induces rumen dysbiosis, leading to LPS translocation and systemic immune activation. This study investigated the effects of a high-grain diet and intramammary LPS challenge on the milk microbiome of dairy cows. Cows were first fed a baseline control diet (day-7 to d-1; CON; 40% grain; n = 18). On d1, 12 cows were switched to a SARA diet (60% grain). On d30, six SARA (SARA_LPS) and the six CON cows (CON_LPS) were challenged intramammarily with LPS, while the other six SARA cows received a placebo (SARA_PLA). No CON_PLA group was enrolled. Milk samples were collected on d-2 (before feeding challenge), d30 (after feeding challenge; before LPS), and d32 (after LPS), and analysed using 16S rRNA gene amplicon sequencing and qPCR. Results During the feeding phases, more genera (70.1%) increased in CON than SARA cows, whereas more (65.3%) genera decreased their relative abundance in SARA compared to CON (p < 0.001). This decline persisted in SARA_PLA cows, with more genera (62.4%) decreasing their abundance (p < 0.001). However, LPS injection reversed the trend of the feeding effect, with more genera (79.3%) increasing in SARA_LPS cows in comparison to the other two groups, while more genera (85.5%) decreased in CON_LPS (p < 0.001) in comparison to the other two groups. Alpha diversity correlated positively with bacterial cell equivalents. Of all genera, 22.1% correlated negatively with milk amyloid A (MAA), which increased post-LPS injection, 21.7% positively with lactose, and 13.4% positively with milk urea. SCC showed significant differences in beta-diversity, but no distinct visual clustering nor many correlations. Conclusion The microbial dynamics suggest that high-grain diet and the LPS injection influence the milk bacterial community. More taxa correlated with MAA than with SCC, suggesting that MAA may better reflect immune-microbial interactions in milk. A roughage-rich diet promoted higher microbial abundance, whereas high-grain feeding reduced abundance over the timespan of 30 days. Intramammary LPS challenge decreased absolute abundance in CON but increased it in SARA cows, suggesting a diet-dependent immune modulation of the mammary environment. These findings indicate that mammary gland epithelial integrity and immune mediators jointly shape the milk microbiome under metabolic and inflammatory stress. PDFDocument eng 2026-08-12T08:48:12.139604509Z 2026 Udder Health High-grain Feeding Mastitis Somatic Cell Count Milk Amyloid A Gut-mammary Gland Axis 6043818 b application/pdf http://creativecommons.org/licenses/by/4.0/