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Mastitis is one of the major problems for the productivity of dairy cows and its classifications have usually been based on milk somatic cell counts (SCCs). In this study, we investigated the differences in milk production, rumen fermentation parameters, and diversity and composition of rumen and hindgut bacteria in cows with similar SCCs with the aim to identify whether they can be potential microbial biomarkers to improve the diagnostics of mastitis. A total of 20 dairy cows with SCCs over 500 × 10 3  cells/mL in milk but without clinical symptoms of mastitis were selected in this study. Random forest modeling revealed that  Erysipelotrichaceae  UCG 004 and the [ Eubacterium ]  xylanophilum  group in the rumen, as well as the Family XIII AD3011 group and  Bacteroides  in the hindgut, were the most influential candidates as key bacterial markers for differentiating “true” mastitis from cows with high SCCs. Mastitis statuses of 334 dairy cows were evaluated, and 96 in 101 cows with high SCCs were defined as healthy rather than mastitis according to the rumen bacteria. Our findings suggested that bacteria in the rumen and hindgut can be a new approach and provide an opportunity to reduce common errors in the detection of mastitis.

Rumen and Hindgut Bacteria Are Potential Indicators for Mastitis of Mid-Lactating Holstein Dairy Cows