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Differentiation of Staphylococci and Streptococci in Milk of Dairy Cows based on Quantitative Proteome

BACKGROUND: Bacteria from Staphylococcus and Streptococcus genera are frequent causes of mastitis in dairy cows. We hypothesized that the quantitative changes in the milk proteome differed depending on presence of Staphylococci or Streptococci. METHODS: We analyzed 118 milk samples from cows with subclinical mastitis, clinical mastitis, and positive controls (group with normal somatic cell count and positive bacterial culture), classified into the groups with Staphylococci (SPH, N=51) or Streptococci (STP, N=67). Sample preparation included proteins՚ reduction, alkylation, precipitation using acetone, digestion with trypsin, and labeling with tandem mass tags. The liquid chromatography-tandem mass spectrometry analysis included separation using the linear gradient and scanning in the positive ion mode using the data-dependent analysis Top8 method, on a modular system consisting of Ultimate 3000 RSLCnano system (Dionex®) coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific®). The proteome Discoverer® software was employed for proteins՚ identification and relative quantitation following the SEQUEST algorithm. Further, the data were analyzed with the R statistical package and MedCalc® software. RESULTS: Among the 386 identified proteins, 20 had different abundance in SPF and STP groups. Eleven proteins had a higher abundance in the SPF group. They included apolipoprotein E, a regulator of protein kinase C, regulators of the coagulation cascade, complement activity, antigen binding, inflammatory response, etc. Among nine proteins that were more abundant in the STP group, the most important were: apolipoprotein A1, coagulation cascade proteins, and proteins involved in cell proliferation, ion transport, homeostasis of thiol components, antigen processing, and presentation. Furthermore, three proteins showed the highest potential to differentiate between the SPF and STP groups with satisfactory accuracy. Those were protein kinase C-binding protein NELL2, thrombospondin-1, and complement factor I, which all had higher abundance in the SPF group. CONCLUSION: The milk proteome pattern differs depending on bacterial culture in milk. Further studies will additionally address pathophysiological changes and clinical utility of the observed differences.

milk, Staphylococci, Streptococci, proteomics

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