Microbial Shifts in Subclinical Mastitis Milk of Tropical Dairy Cows Revealed by Nanopore 16S Sequencing

Subclinical mastitis remains a major challenge in dairy cattle health, particularly in smallholder farms within tropical regions. This study aimed to profile the milk microbiota of healthy dairy cows and those with early-stage subclinical mastitis (score 1) using full-length 16S rRNA sequencing via Oxford Nanopore technology, while exploring the ecological flexibility of low-abundance taxa. Milk samples were aseptically collected from healthy cows and cows diagnosed with subclinical mastitis (score 1) based on the California Mastitis Test. 16S rRNA gene sequencing was performed, followed by taxonomic classification and statistical analyses. Statistical comparison of bacterial diversity between healthy and subclinical mastitis (CMT score 1) milk at the genus and species levels was conducted using Levene’s test and independent t-tests. Bartonella and Caulobacter were dominant in healthy milk, whereas Epilithonimonas and Phyllobacterium were more abundant in mastitic samples. Species such as Epilithonimonas vandammei and Phyllobacterium pellucidum were identified as potential early-stage biomarkers. Certain taxa, including Rothia and Sutterella, demonstrated ecological plasticity by being present in both healthy and mastitic milk conditions. Early-stage subclinical mastitis (score 1) was associated with qualitative changes in milk microbiota, with key low-abundance taxa showing potential as early indicators. These findings offer new insights into mastitis pathogenesis and support the development of microbiota-based early detection strategies for smallholder dairy farming systems.