ABSTRACT: In a recent study, a teat disinfectant based on a nisin-producing Lactococcus cremoris displayed comparable efficacy to a conventional iodophor disinfectant in preventing dairy cow mastitis when implemented in the dairy cow milking routine. Here, we carried out a longitudinal study for an in-depth investigation of the milk proteome. Four groups of matched quarter milk with low and high somatic cell count (SCC) collected at the beginning (T0) and at the end (TF) of the trial were selected for analysis, for a total of 28 low SCC (LSCC) and 12 high SCC (HSCC) samples. The milk was delipidated, digested into peptides, and subjected to a proteomic analysis workflow entailing NanoHPLC separation and tandem mass spectrometry (LC-MS/MS) on an Orbitrap Fusion Tribrid mass spectrometer. The proteins were identified with MaxQuant, the proteomes were compared for identifying differential proteins, and their interaction networks were assessed with STRING. In LSCC milk at T0, 31 were increased or present only in the Lactococcus (L) disinfectant group (from now INCREASED in L) and 59 proteins were decreased in L disinfectant group or present only in the conventional iodophor (C) group (from now DECREASED in L). All were involved in metabolic pathways and were likely related to physiological differences between animals. In LSCC milk at TF, 34 proteins were INCREASED in L and 46 proteins were DECREASED in the L disinfectant group. Most belonged to physiological milk pathways, but 3 antimicrobial proteins were enriched in the L group at the end of the trial. In HSCC milk at T0, 54 proteins were INCREASED in L and 34 DECREASED in L. Most were involved in physiological metabolic pathways; 4 proteins with antimicrobial functions were higher in L at the beginning of the trial. In HSCC milk at TF, 67 proteins were INCREASED in L and 39 DECREASED in L. All the proteins enriched in C belonged to metabolic pathways, while up to 14 proteins enriched in L were involved in antibiotic, antimicrobial, and immune defense pathways. Cathelicidins were among the most prominent differential proteins with antimicrobial functions. At T0, cathelicidins were generally higher in HSCC than LSCC milk of both experimental groups, as expected. At TF, they were increased or unique only in the Lactococcus group, in both LSCC and HSCC milk. Western immunoblotting validation confirmed the proteomic findings. On the other hand, enzymatic assessment of n-acetyl-glucosaminidase (NAGase) did not highlight significant differences between the C and L groups, neither for LSCC nor for HSCC milk. In conclusion, the two disinfectants did not induce relevant functional changes in the milk proteome of healthy animals, except for three increased antimicrobial proteins in the Lactococcus group. On the other hand, in animals with an underlying mammary gland inflammation, the milk proteome of the Lactococcus group showed increased levels of several proteins with antibiotic, antimicrobial and immune defense functions. Among other factors, the selective immunostimulatory action of nisin, the antimicrobial effector of L. cremoris, might play a role in this observation.
Simona Nonnis 
