Application of culture, PCR, and PacBio sequencing for determination of microbial composition of milk from subclinical mastitis dairy cows of smallholder farms

Mastitis is a cow disease usually signalized by irritation, swelling, and soreness of the udder. It is characterized by physical, chemical, and biological changes in the udder and milk. The aim of this study was to detect and characterize pathogens causing subclinical mastitis (SCM) from the milk of dairy cows of small-scale farmers through culture and molecular techniques. Milk was collected from 32 cows belonging to 8 small-scale farmers around Harrismith District, South Africa. The results showed that screening of SCM by California mastitis test and somatic cell counts (SCC) was 21.87 and 25%, respectively. Culture methods revealed the presence of Staphylococcus aureus at 93% followed by Streptococci spp. and Escherichia coli at 36.4 and 13.3%, respectively. The PCR could only detect E. coli, while single-molecule real-time sequencing showed a total of 2 phyla, 5 families, 7 genera, and 131 species. Clostridiaceae was the most abundant family, while Romboutsia was the most abundant genus followed by Turicibacter spp. The present study has documented the occurrence of SCM causing pathogens in milk collected from cows of small-scale farmers in Harrismith, indicating that SCM may be present at higher levels than expected.