phaidra.vetmeduni.ac.at o:5410 DOI 10.1186/s42523-025-00499-5 Dynamics of the milk microbial community during subacute ruminal acidosis with or without intramammary lipopolysaccharide challenge in dairy cows 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. eng Udder Health High-grain Feeding Mastitis Somatic Cell Count Milk Amyloid A Gut-mammary Gland Axis 2026-08-12T08:48:12.361Z LOMv1.0 Author BEGIN:VCARD VERSION:3.0 N:Neubauer;Viktoria; FN:Viktoria Neubauer X-ORCID:https://orcid.org/0000-0002-4312-5781 END:VCARD BEGIN:VCARD VERSION:3.0 N:Aditya;Siska; FN:Siska Aditya END:VCARD BEGIN:VCARD VERSION:3.0 N:Quijada;Narciso M.; FN:Narciso M. Quijada END:VCARD BEGIN:VCARD VERSION:3.0 N:Wetzels;Stefanie Urimare; FN:Stefanie Urimare Wetzels END:VCARD BEGIN:VCARD VERSION:3.0 N:Dzieciol;Monika; FN:Monika Dzieciol X-ORCID:https://orcid.org/0000-0002-0972-6241 END:VCARD BEGIN:VCARD VERSION:3.0 N:Pourazad;Poulad; FN:Poulad Pourazad END:VCARD BEGIN:VCARD VERSION:3.0 N:Zebeli;Qendrim; FN:Qendrim Zebeli X-ORCID:https://orcid.org/0000-0001-5188-9004 END:VCARD BEGIN:VCARD VERSION:3.0 N:Selberherr;Evelyne; FN:Evelyne Selberherr X-ORCID:https://orcid.org/0000-0002-2253-5247 END:VCARD https://w3id.org/kim/hcrt/scheme https://w3id.org/kim/hcrt/text Textdokument Text LOMv1.0 yes https://creativecommons.org/licenses/by/4.0 application/pdf 6043818 https://phaidra.vetmeduni.ac.at/o:5410 https://phaidra.vetmeduni.ac.at/api/object/o:5410/thumbnail LOMv1.0 discipline