Project description:BackgroundDairy cow milking practices require cleaning and disinfection of the teat skin before and after milking to ensure the safety and quality of milk and prevent intramammary infections. Antimicrobial proteins of natural origin can be valuable alternatives to traditional disinfectants. In a recent field trial, we demonstrated that a teat dip based on a nisin A-producing Lactococcus cremoris (L) had comparable efficacy to conventional iodophor dip (C) in preventing dairy cow mastitis. Here, we present the differential shotgun proteomics investigation of the milk collected during the trial.MethodsFour groups of quarter milk samples with low (LSCC) and high somatic cell count (HSCC) collected at the beginning (T0) and end (TF) of the trial were analyzed for a total of 28 LSCC (14 LSCC T0 and 14 LSCC TF) and 12 HSCC (6 HSCC T0 and 6 HSCC TF) samples. Milk proteins were digested into peptides, separated by nanoHPLC, and analyzed by tandem mass spectrometry (LC-MS/MS) on an Orbitrap Fusion Tribrid mass spectrometer. The proteins were identified with MaxQuant and interaction networks of the differential proteins were investigated with STRING. The proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD045030.ResultsIn healthy milk (LSCC), we detected 90 and 80 differential proteins at T0 and TF, respectively. At TF, the Lactococcus group showed higher levels of antimicrobial proteins. In mastitis milk (HSCC), we detected 88 and 106 differential proteins at T0 and TF, respectively. In the Lactococcus group, 14 proteins with antimicrobial and immune defense functions were enriched at TF vs. 4 proteins at T0. Cathelicidins were among the most relevant enriched proteins. Western immunoblotting validation confirmed the differential abundance.ConclusionsAt T0, the proteomic differences observed in healthy milk of the two groups were most likely dependent on physiological variation. On the other hand, antimicrobial and immune defense functions were higher in the milk of cows with mammary gland inflammation of the Lactococcus-treated group. Among other factors, the immunostimulatory action of nisin A might be considered as a contributor.

Project description:In pasture-based systems, changes in dairy herd habitat due to seasonality results in the exposure of animals to different environmental niches. These niches contain distinct microbial communities that may be transferred to raw milk, with potentially important food quality and safety implications for milk producers. It is postulated that the extent to which these microorganisms are transferred could be limited by the inclusion of a teat preparation step prior to milking. High-throughput sequencing on a variety of microbial niches on farms was used to study the patterns of microbial movement through the dairy production chain and, in the process, to investigate the impact of seasonal housing and the inclusion/exclusion of a teat preparation regime on the raw milk microbiota from the same herd over two sampling periods, i.e., indoor and outdoor. Beta diversity and network analyses showed that environmental and milk microbiotas separated depending on whether they were sourced from an indoor or outdoor environment. Within these respective habitats, similarities between the milk microbiota and that of teat swab samples and, to a lesser extent, fecal samples were apparent. Indeed, SourceTracker identified the teat surface as the most significant source of contamination, with herd feces being the next most prevalent source of contamination. In milk from cows grazing outdoors, teat prep significantly increased the numbers of total bacteria present. In summary, sequence-based microbiota analysis identified possible sources of raw milk contamination and highlighted the influence of environment and farm management practices on the raw milk microbiota. The composition of the raw milk microbiota is an important consideration from both a spoilage perspective and a food safety perspective and has implications for milk targeted for direct consumption and for downstream processing. Factors that influence contamination have been examined previously, primarily through the use of culture-based techniques. We describe here the extensive application of high-throughput DNA sequencing technologies to study the relationship between the milk production environment and the raw milk microbiota. The results show that the environment in which the herd was kept was the primary driver of the composition of the milk microbiota composition.

Project description:BackgroundBovine mastitis is a major challenge in dairy farms. Since the agents commonly used for pre- and post-dipping can affect the udder health by modifying milk microbiota, alternative products are needed. This study aimed to evaluate the effect of the use of pre- and post-dipping formulations containing the fermented broth of Nisin A-producing Lactococcus cremoris FT27 strain (treated group, TR) on the abundance and biodiversity of milk microbiota as compared to iodine-based commercial disinfectants (control group, CTR) during a three-month trial. The experiment was conducted on 20 dairy cows, divided into two groups (CTR and TR) of 10 lactating cows each. Milk samples were collected from two selected healthy quarters of each cow at 3 time-points. Microbial communities were investigated by cultural and sequence-based methods, and analyzed through bioinformatic and statistical approaches.ResultsClear differences in bacterial community composition were observed among groups, with higher species richness in TR, especially of Staphylococcus, Enterococcus, Lactococcus, and Streptococcus genera. The microbiota was dominated by Firmicutes, followed by Actinobacteriota, Proteobacteria, and Bacteroidota. Staphylococcaceae family was significantly higher in TR (p < 0.009), whereas Carnobacteriaceae, Mycobacteriaceae, and Pseudomonadaceae were significantly lower (p = 0.005, p = 0.001, and p = 0.040, respectively). CTR had considerably higher abundances of the genera Alkalibacterium (p = 0.011), Pseudomonas_E (p = 0.045), Corynebacterium (p = 0.004), and Alloiococcus (p = 0.004), and lower abundances of Staphylococcus (p < 0.009). Milk microbiota changed noticeably during the experimental period, regardless of treatment. A significant decrease was observed in both groups for Firmicutes_A phylum, with an increment in Actinobacteriota phylum, Propionibacteriaceae family, and Cutibacterium genus. Streptococcaceae significantly decreased in CTR (p = 0.013) and rose in TR (p = 0.001). Several differences were observed between the two groups during the experimental period. Streptococcus genus almost disappeared in CTR (p = 0.013), whereas it significantly increased in TR (p = 0.001). Three and twelve enriched groups were significantly identified respectively in CTR and TR using LEfSe.ConclusionsThe use of Nisin A-based teat dip formulations could be linked to greater microbial diversity compared to commercial products. Despite the influence of seasonality, the experimental formulations maintained higher milk biodiversity, suggesting that lactic acid bacteria metabolites prevent alterations in the milk microbiota.

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