Exploring the Milk Microbiota of Healthy and Mastitic Nili Ravi Buffalo Using 16S rRNA Gene Base Metagenomic Analysis

SIMPLE SUMMARY: Mastitis is a prevalent issue worldwide among lactating animals. It continues to be a serious problem since it has an economic impact on farmers due to direct and indirect losses. The multifactorial nature of this disease, coupled with the limitations of classical culture-based methods to identify uncultivable bacteria under normal laboratory conditions, exacerbates the situation. In the current study, milk samples from the well-known buffalo breed (Nili Ravi) were tested to explore bacterial communities associated with different udder health statuses using the 16S rRNA gene-based metagenomics technique. The bacterial communities in buffaloes with different udder health statuses, such as healthy, clinical mastitis, and subclinical mastitis, exhibited varied abundance at different taxonomic levels. Additionally, the study concluded that bacterial diversity in milk samples decreases in animals suffering from clinical mastitis compared to those with a healthy or subclinical mastitis status. These findings will contribute to our understanding of bacterial diversity’s role in mastitis development. ABSTRACT: The Nili Ravi, a buffalo breed from Pakistan, significantly contributes to the dairy industry. Mastitis is one of the major challenges affecting milk production in this breed. The objective of the current study was to identify the bacterial communities and diversity in healthy and mastitic milk of this breed. Milk samples (n = 14) were collected from Nili Ravi buffaloes with different udder health statuses, i.e., healthy (5), subclinical mastitis (4), and clinical mastitis (5). The DNAs were extracted, subjected to partial amplification of 16S rDNA (V3 and V4 regions), and sequenced using the Illumina platform. The results revealed variations in the bacterial communities in the milk of animals with different udder health statuses. Proteobacteria was the predominant phylum in the healthy group, while clinical and subclinical mastitis milk had a higher abundance of Firmicutes. Dominant bacterial genera in the healthy group were Streptococcus (11.60%), Herbaspirillum (7.65%), and Staphylococcus (4.70%), whereas the clinical mastitis group was dominated by Streptococcus (33.96%), Staphylococcus (7.87%), and Corynebacterium (2.68%), and the subclinical mastitis group was dominated by Bacillus (15.70%), Corynebacterium (6.70%), and Staphylococcus (6.58%). Assignment of operational taxonomic units at the species level resulted in most species being assigned to uncultured or unknown bacteria or remaining unassigned. Alpha diversity indices indicated lower microbial diversity in the clinical mastitis group, while beta diversity indices showed a scattered pattern of sample clustering in PCA plots among different groups. It is concluded that bacterial diversity in the milk of Nili Ravi buffaloes suffering from clinical mastitis is lower compared to healthy and subclinical mastitis cases. It is concluded that the variations in the microbiota of healthy and mastitic milk may be further investigated and exploited as signature microbes associated with the udder health status of Nili Ravi buffalo.