Peptidomic changes in the milk of water buffaloes (Bubalus bubalis) with intramammary infection by non-aureus staphylococci

Abstract

Mastitis by non-aureus staphylococci (NAS) is a significant issue in dairy buffalo farming. In a herd with subclinical NAS mastitis, we identified Staphylococcus microti as the predominant species. To assess milk protein integrity and investigate potential disease markers, we characterized 12 NAS-positive and 12 healthy quarter milk samples by shotgun peptidomics combining peptide enrichment and high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS). We observed significant changes in the milk peptidome. Out of 789 total peptides identified in each group, 49 and 44 were unique or increased in NAS-positive and healthy milk, respectively. In NAS-positive milk, the differential peptides belonged mainly to caseins, followed by milk fat globule membrane proteins (MFGMP) and by the immune defense/antimicrobial proteins osteopontin, lactoperoxidase, and serum amyloid A. In healthy milk, these belonged mainly to MFGMP, followed by caseins. In terms of abundance, peptides from MFGMP and immune defense protein were higher in NAS-positive milk, while peptides from caseins were higher in healthy milk. These findings highlight the impact of NAS on buffalo milk quality and mammary gland health, even when clinical signs are not evident, and underscore the need for clarifying the epidemiology and relevance of the different NAS species in this dairy ruminant.

Figure 1

Shotgun label-free quantitative peptidomic analysis. (A) Overview of the protocols applied for the analysis of peptides in NAS-positive and healthy milk samples (B) Venn diagram of all the peptides identified in milk samples from healthy and NAS-positive buffaloes. Peptides were considered differentially abundant if they were present only in NAS-positiver or Control milk or showed significant Welch t test difference (cut-off at 1% permutation-based False Discovery Rate).

Figure 2

Distribution of the differential peptides identified in NAS-positive (red) and healthy milk (blue) according to the cell location/function of the originating protein. The histograms display the number (A) and the abundance (B) of peptides in NAS-positive and healthy milk samples, respectively.

Figure 3

Relative amino acid frequencies at the C-terminus. The figure illustrates the relative distribution of C-terminal amino acids in unique and significantly differential peptides found in NAS-positive (red) and healthy milk (blue), respectively.