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

Abstract

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.

Development of a lateral flow device for rapid simultaneous multiple detections of some common bacterial causes of bovine mastitis

“…Laboratory diagnosis of bovine mastitis through bacteriological isolation and identification of the causative microorganisms is the gold master test. However, it is expensive, time-consuming, and requires a fully equipped laboratory and experienced staff [ 27 ] Moreover, the molecular diagnosis of bovine mastitis using PCR is sensitive and specific for the diagnosis of clinical and subclinical mastitis and could be used in the detection of pathogens in milk samples at the species level in few hours [ 28 , 29 ]. However, again, it is expensive, not suitable as a field test, and like the bacteriological examination, requires a specially equipped laboratory.…”

Metagenomic analysis of milk microbiota in the bovine subclinical mastitis

“…Bovine intramammary inflammation represents a worldwide burden causing serious repercussions not only on the health of dairy herds, but also on milk productivity and quality (15,46). To limit the spread of this disease, and especially of its silent form, i.e., subclinical mastitis, whose containment is difficult due to the lack of evident symptoms and its high incidence rate, the identification of SM microbial causative agents is of crucial significance (12,(47)(48)(49).…”

“…Indeed, most of publicly available metagenomic studies only employed 16S rRNA gene microbial profiling-based sequencing, thus preventing an accurate and complete characterization of the bovine milk microbiota associated to SM down to the species level (3,6,7,11,12). At the same time, studies limited to culture-dependent investigations, despite being able to identify the presence of underrepresented pathogenic microorganisms in subclinical bovine milk whose detection can escape metagenomics due to the intrinsic limit of this molecular approach (13), do not allow to obtain an accurate overview of how the milk microbiota can change during SM (14)(15)(16).…”

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

“…Mastitis is regarded as a significant illness with a global impact on the dairy business, including medicine and disease management expenses, death among sick animals, and recurrence costs [1]. The most common kind of mastitis, subclinical mastitis, is an asymptomatic form of intramammary inflammation that affects 20-50% of cows in a particular herd.…”