Characterisation of Milk Microbiota from Subclinical Mastitis and Apparently Healthy Dairy Cattle in Free State Province, South Africa

SIMPLE SUMMARY: Mastitis is an inflammation of the mammary gland caused by mechanical, physical, chemical, and biological causes. The identification of mastitis causative agents was accomplished using microbiological culture and DNA techniques, a common method in veterinary medicine. Moreover, significant advancements in high throughput next generation sequencing (NGS) and bioinformatics techniques have facilitated the transition from culture-based methods to genomic sequence-based characterisation of the microbiome associated with mastitis infection in both humans and animals. Hence, this study utilised 16S metagenomics to understand the taxonomic profiles of both subclinical mastitis (SCM) and apparently healthy (non-SCM) dairy cow milk of small-scale farmers. Overall, the analysis indicated a total of 95 phyla, 33 classes, 884 orders, 124 families, 202 genera, and 119 bacterial species. The data analysis clearly indicates that the microbiome composition in SCM and non-SCM cows is considerably different. ABSTRACT: Bovine mastitis is an inflammation of the udder tissue of the mammary gland brought on by microbial infections or physical damage. It is characterised by physical, chemical, and biological changes in the udder and milk. While several different bacterial species have been identified as causative agents of mastitis, many subclinical mastitis (SCM) cases remain culture-negative. The aim of this study was to characterise milk microbiota from SCM and apparently healthy dairy cows (non-SCM) by 16S rRNA sequencing. Alpha-diversity metrics showed significant differences between SCM cows and non-SCM counterparts. The beta-diversity metrics in the principal coordinate analysis significantly clustered samples by type (PERMANOVA test, p < 0.05), while non-metric dimensional scaling did not (PERMANOVA test, p = 0.07). The overall analysis indicated a total of 95 phyla, 33 classes, 82 orders, 124 families, 202 genera, and 119 bacterial species. Four phyla, namely Actinobacteriota, Bacteroidota, Firmicutes, and Proteobacteria collectively accounted for more than 97% of all sequencing reads from SCM and non-SCM cow samples. The most abundant bacterial classes were Actinobacteria, Bacilli, Bacteroidia, Clostridia, and Gammaproteobacteria in non-SCM cow samples, whilst SCM cow samples were mainly composed of Actinobacteria, Alphaproteobacteria, Bacilli, Clostridia, and Gammaproteobacteria. Dominant bacterial species in non-SCM cow samples were Anthropi spp., Pseudomonas azotoformans, P. fragi, Acinetobacter guillouiae, Enterococcus italicus, Lactococcus lactis, whilst P. azotoformans, Mycobacterium bovis, P. fragi, Acinetobacter guillouiae, and P. koreensis were dominant in the SCM cow samples. The current study found differences in bacterial species between SCM and non-SCM cow milk; hence, the need for detailed epidemiological studies.