Exploratory characterization of the milk bacterial microbiome in subclinical mastitis at CMT scores 2 and 3 in smallholder dairy farms using full-length nanopore 16S rRNA sequencing

Subclinical mastitis and bacterial taxonomic profile

Abstract

This exploratory study characterized the taxonomic profiles of bacterial microbiota associated with CMT scores 2 and 3 in Holstein-Friesian dairy cows from smallholder farms in Batu City, East Java, Indonesia. A total of 126 cows from 29 smallholder farms were screened using the California Mastitis Test (CMT), and 27 cows had CMT scores of 2 and 3. One representative milk sample was selected for each condition: SP3 for CMT score 2 and SP4 for CMT score 3. Full-length 16S rRNA gene sequencing was performed using Oxford Nanopore Technology (ONT), and taxonomic classification was conducted using Centrifuge against the NCBI 16S RefSeq database. The SP3 sample yielded 99,600 high-quality reads and showed a Streptococcus-dominated taxonomic profile, with Bacillota as the predominant phylum and the S. dysgalactiae group as the dominant species-level taxonomic assignment. Corynebacterium and Clostridium were also detected. The SP4 sample yielded 98,300 high-quality reads and exhibited a highly skewed taxonomic profile, with S. agalactiae as the predominant species-level taxonomic assignment. These observations indicate distinct taxonomic profiles between the two representative samples, but they should be interpreted descriptively because only one sample was analyzed for each CMT condition. The findings provide an exploratory basis for further investigation of milk microbiome patterns associated with subclinical mastitis severity using full-length Nanopore 16S rRNA sequencing. Larger studies with adequate biological replication and independent species confirmation are required to validate these taxonomic patterns and assess their potential diagnostic relevance.