Characterizing the Milk Microbiome in Subclinical Mastitis: A Pilot 16S rRNA‐Based Study in Cattle and Water Buffalo

BACKGROUND: In the dairy sector of Bangladesh, subclinical mastitis (SCM) is a substantial and frequently undiagnosed challenge, with reported prevalence rates of 60%-77% in cattle and approximately 52% in buffaloes. Due to its complex characteristics and progressive development, efficient diagnosis and management are essential for enhancing dairy productivity. OBJECTIVES: This pilot study employed 16S rRNA amplicon sequencing using Oxford Nanopore's MinION to investigate the milk microbiota of healthy and mastitic cattle and buffalo. RESULTS: A total of 423 clustered nucleotide sequences were identified in the samples, indicating significant taxonomic diversity: 11 phyla, 26 classes, 58 orders, 120 families and 272 genera. Distinct phylum-level patterns were observed, with Firmicutes predominating in healthy milk and a relative increase in Proteobacteria and Actinobacteriota in mastitic samples. At the genus level, Streptococcus and Lactococcus were predominant in mastitic samples, whereas Staphylococcus and Lactococcus were more prevalent in healthy milk. The results indicate that although overall microbial diversity was relatively consistent across groups, mastitis correlated with alterations in bacterial community composition, with notable differences between cattle and buffalo. CONCLUSION: This study suggests a potential association between SCM and microbial shifts; however, microbiome profiling cannot yet be recommended for diagnostic application. Clinical applicability requires validation in large-scale studies with individual-level sampling.