Microbiome

Decoding the milk microbiome-host interaction in mastitis susceptible and resistant Holstein cows with subclinical intramammary infection

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Abstract

This study aimed to longitudinally characterize the milk microbiome of mastitis-resistant and mastitis-susceptible Holstein cows with subclinical intramammary infection (sIMI), to explore host genetics-microbiome interactions in mastitis. Specifically, single-quarter milk samples were aseptically collected from 200 genetically mastitis-resistant and 200 genetically mastitis-susceptible Holstein cows and subjected to an initial microbiological screening (T0). Based on results, animals were classified into two groups: those with at least one quarter positive for mastitis-causing pathogens, i.e. Staphylococcus aureus, Streptococcus uberis, or Streptococcus dysgalactiae (Pos), and those negative in all quarters (Neg). Among these, 86 animals were selected for the longitudinal evaluation and sampled again 2 and 5 weeks after T0 (T1 and T2, respectively). Milk microbial DNA was extracted using two different methods (commercial column-based kit and magnetic bead-based automated extractor) and sequenced via a shotgun approach. Milk metagenome was analyzed in an initial batch of 32 samples (16 collected at T1 and 16 at T2) extracted with the column-based method. After raw data trimming and host DNA depletion, sequences were analyzed to profile the composition of microbial communities using Kraken2 and Bracken software. Analyses of diversity indices among different the microbiological conditions were performed on samples collected at T1, as this time point was expected to reflect the peak of microbiome alterations following sIMI.A mean of 32 ± 14 % reads were classified, identifying 1,362 different species across all samples. Among them, 905 species were exclusive to the Neg samples (n = 25), 20 to the Pos samples (n = 7), and 406 were shared, highlighting a strong reduction in microbial diversity and a loss of community complexity associated with sIMI. The most abundant classified species in Pos samples were Staphylococcus aureus, up to 70 %, followed by Escherichia coli, whereas negative samples were dominated by Escherichia coli and Corynebacterium casei. Six to ten species were among the top-ten species in both groups, representing the core microbiota. Despite these compositional differences, no significant differences in α-diversity indices were observed at T1 (12 Neg and 4 Pos), while β-diversity (Bray-Curtis) analysis revealed a significant separation between Pos and Neg samples (P Future analyses will focus on differential abundance testing, including the mastitis-resistant and -susceptible status. In addition, metagenome-assembled genome reconstruction will be performed to classify the unassigned reads, including searching for antibiotic resistance genes. Overall, this work will provide a deeper understanding of milk microbiome dynamics and their relationship with host genetics during subclinical intramammary infection.Acknowledgements: MASTITOMIC project funded by the European Union Next-Generation EU

Keywords: 2026

How to Cite:

Vanzin, A., Giannotta, G., Bisutti, V., Secchi, G., Giannuzzi, D., Zappaterra, M., Barberio, A., Cassandro, M., Marusi, M., Finocchiaro, R., Cecchinato, A. & Pegolo, S., (2026) “Decoding the milk microbiome-host interaction in mastitis susceptible and resistant Holstein cows with subclinical intramammary infection”, World Congress on Genetics Applied to Livestock Production Digital Archive 2026(1): 2286365. doi: https://doi.org/10.31274/wcgalp.23960

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