Hoof, Vaginal and Milk Microbiota Alterations in Dairy Cows with Foot Rot

Foot rot is a major infectious cause of lameness in dairy cows and can severely compromise production performance and animal welfare. However, it remains unclear whether foot rot is accompanied by microbiota alterations beyond the hoof, particularly in the vagina and milk. We conducted a case-control study of 24 Holstein cows (12 with clinical foot rot and 12 healthy controls). Hoof swabs, milk, and vaginal swabs were collected and analyzed using 16S rRNA gene amplicon sequencing, alongside quantitative PCR. Microbial community composition differed between cows with foot rot and healthy controls across hoof (R² = 0.26), vaginal (R² = 0.072), and milk (R² = 0.16) samples (Bray-Curtis, PERMANOVA, <i>p</i> < 0.001). Simpson diversity was lower in the hoof and vaginal microbiota (<i>p</i> < 0.05) but not in milk (<i>p</i> = 0.13). Differential abundance analysis identified enrichment of <i>Fusobacterium</i> in hoof and vaginal samples (<i>p</i> < 0.001), and absolute qPCR confirmed a higher vaginal load of <i>Fusobacterium necrophorum</i> in cows with foot rot (<i>p</i> < 0.05). Milk samples from foot rot cows showed enrichment of <i>Clostridium</i> and <i>Porphyromonas</i>. Co-occurrence network analysis revealed reduced microbial connectivity in foot rot-affected cows (average degree: 4.98 vs. 6.70) and increased network fragmentation (14 vs. 9 modules), suggesting lower ecosystem stability. Overall, foot rot was associated with microbiota shifts across the hoof, vagina, and milk, highlighting the potential value of integrated management strategies that consider extra-hoof microbial alterations.