Alessandro Toscano

Understanding the relationship between microbial community alterations and disease can provide valuable insights for improving diagnostics, prevention, and treatment strategies. This study used 16S rRNA amplicon sequencing to investigate and compare microbial diversity in the milk and feces of Holstein cows with subclinical mastitis caused by Streptococcus agalactiae and Prototheca spp. with that of healthy cows. A bacteriological screening identified 50 Holstein cows reared in a commercial dairy farm and classified into 3 experimental groups: (1) animals negative at the bacteriological examination with no history of subclinical mastitis (HLTH; n = 16), (2) animals positive at the bacteriological examination for either Streptococcus agalactiae (STRP; n = 22) or Prototheca spp. (PRTH; n = 12). The milk microbiota showed significant pathogen-specific alterations, with increased Firmicutes in STRP cows (55.6%) and Cyanobacteria in PRTH cows (17.3%), compared with healthy cows (39.2% and 0.7%, respectively). Alpha diversity (observed amplicon sequence variants, Shannon, and evenness indices) was significantly lower in infected cows, confirming a microbial imbalance. Beta diversity analysis (PERMANOVA) revealed significant differences in microbial composition between healthy and infected cows, but no significant differences in fecal microbiota composition. Differential abundance analysis identified Streptococcus (log₂fold change [FC] = 7.3) as the most enriched taxon in STRP cows and Cyanobacteria (log₂FC = 8.9) as the most enriched in PRTH cows in milk matrix, while Macrococcus caseolyticus was significantly reduced in both infected groups (log₂FC = -4.5). These findings suggest that subclinical mastitis leads to significant shifts in the milk microbiota but does not alter the fecal microbiome, supporting a localized rather than systemic microbial response. This study provides novel insights into the microbial dynamics of subclinical mastitis and potential biomarkers for disease monitoring.

In this study we wanted to investigate the associations between naturally occurring subclinical intramammary infection (IMI) caused by different etiological agents (i.e., Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, and Prototheca spp.), in combination with somatic cell count (SCC), on the detailed milk protein profile measured at the individual mammary gland quarter. An initial bacteriological screening (time 0; T0) conducted on individual composite milk from 450 Holstein cows reared in 3 herds, was performed to identify cows with subclinical IMI. We identified 78 infected animals which were followed up at the quarter level at 2 different sampling times: T1 and T2, 2 and 6 wk after T0, respectively. A total of 529 quarter samples belonging to the previously selected animals were collected at the 2 sampling points and analyzed with a reversed phase HPLC (RP-HPLC) validated method. Specifically, we identified and quantified 4 caseins (CN), namely alpha S1-CN, alpha S2-CN, kappa-CN, and beta-CN, and 3 whey protein fractions, namely beta-lactoglobulin, alpha-lactalbumin, and lactoferrin (LF), which were later expressed both quantitatively (g/L) and qualitatively (as a percentage of the total milk nitrogen content, % N). Data were analyzed with a hierarchical linear mixed model with the following fixed effects: days in milk (DIM), parity, herd, SCC, bacteriological status (BACT), and the SCC x BACT interaction. The random effect of individual cow, nested within herd, DIM and parity was used as the error term for the latter effects. Both IMI (i.e., BACT) and SCC significantly reduced the proportion of beta-CN and alpha S1-CN, ascribed to the increased activity of both milk endogenous and microbial proteases. Less evident alterations were found for whey proteins, except for LF, which being a glycoprotein with direct and undirect antimicrobial activity, increased both with IMI and SCC, suggest ing its involvement in the modulation of both the innate and adaptive immune response. Finally, increasing SCC in the positive samples was associated with a more marked reduction of total caseins at T1, and alpha S1-CN at T2, suggesting a synergic effect of infection and inflammation, more evident at high SCC. In conclusion, our work helps clarify the behavior of protein fractions at quarter level in animals having subclinical IMI. The inflammation status driven by the increase in SCC, rather the infection, was associated with the most significant changes, suggesting that the activity of endogenous proteolytic enzymes related to the onset of inflammation might have a pivotal role in directing the alteration of the milk protein profile.

The considerable increase in the production capacity of individual cows owing to both selective breeding and innovations in the dairy sector has posed challenges to management practices in terms of maintaining the nutritional and metabolic health status of dairy cows. In this observational study, we investigated the associations between milk yield, composition, and technological traits and a set of 21 blood biomarkers related to energy metabolism, liver function or hepatic damage, oxidative stress, and inflammation or innate immunity in a population of 1,369 high-yielding Holstein-Friesian dairy cows. The milk traits investigated in this study included 4 production traits (milk yield, fat yield, protein yield, daily milk energy output), 5 traits related to milk composition (fat, protein, casein, and lactose percentages and urea), 11 milk technological traits (5 milk coagulation properties and 6 curd-firming traits). All milk traits (i.e., production, composition, and technological traits) were analyzed according to a linear mixed model that included the days in milk, the parity order, and the blood metabolites (tested one at a time) as fixed effects and the herd and date of sampling as random effects. Our findings revealed that milk yield and daily milk energy output were positively and linearly associated with total cholesterol, nonesterified fatty acids, urea, aspartate aminotransferase, γ-glutamyl transferase, total bilirubin, albumin, and ferric-reducing antioxidant power, whereas they were negatively associated with glucose, creatinine, alkaline phosphatase, total reactive oxygen metabolites, and proinflammatory proteins (ceruloplasmin, haptoglobin, and myeloperoxidase). Regarding composition traits, the protein percentage was negatively associated with nonesterified fatty acids and β-hydroxybutyrate (BHB), while the fat percentage was positively associated with BHB, and negatively associated with paraoxonase. Moreover, we found that the lactose percentage increased with increasing cholesterol and albumin and decreased with increasing ceruloplasmin, haptoglobin, and myeloperoxidase. Milk urea increased with an increase in cholesterol, blood urea, nonesterified fatty acids, and BHB, and decreased with an increase in proinflammatory proteins. Finally, no association was found between the blood metabolites and milk coagulation properties and curd-firming traits. In conclusion, this study showed that variations in blood metabolites had strong associations with milk productivity traits, the lactose percentage, and milk urea, but no relationships with technological traits of milk. Specifically, increasing levels of proinflammatory and oxidative stress metabolites, such as ceruloplasmin, haptoglobin, myeloperoxidase, and total reactive oxygen metabolites, were shown to be associated with reductions in milk yield, daily milk energy output, lactose percentage, and milk urea. These results highlight the close connection between the metabolic and innate immunity status and production performance. This connection is not limited to specific clinical diseases or to the transition phase but manifests throughout the entire lactation. These outcomes emphasize the importance of identifying cows with subacute inflammatory and oxidative stress as a means of reducing metabolic impairments and avoiding milk fluctuations.

Udder health has a crucial role in sustainable milk production, and various reports have pointed out that changes in udder condition seem to affect milk mineral content. The somatic cell count (SCC) is the most recognized indicator for the determination of udder health status. Recently, a new parameter, the differential somatic cell count (DSCC), has been proposed for a more detailed evaluation of intra-mammary infection patterns. Specifically, the DSCC is the combined proportions of polymorphonuclear neutrophils and lymphocytes (PMN-LYM) on the total SCC, with macrophages (MAC) representing the remainder proportion. In this study, we evaluated the association between DSCC in combination with SCC on a detailed milk mineral profile in 1,013 Holstein Friesian cows reared in 5 herds. An Inductively Coupled Plasma- Optical Emission Spectrometry (ICP-OES) was used to quantify 32 milk mineral elements. Two different linear mixed models were fitted to explore the associations between the milk mineral elements and first, the DSCC combined with SCC, and second, DSCC expressed as the PMN-LYM and MAC counts, obtained by multiplying the proportion of PMN-LYM and MAC by SCC. We observed a significant positive association between SCC and milk Na, S and Fe levels. Differential somatic cell count showed an opposite behavior to the one displayed by SCC, with a negative association with Na and positive association with K milk concentrations. When considering DSCC as count, Na and K showed contrasting behavior when associated to PMN-LYM or MAC counts, with decreasing of Na content and increasing K when associated to increasing PMN-LYM counts, and increasing Na and decreasing K when associated to increasing MAC count. These findings confirmed that an increase in SCC is associated with altered milk Na and K amounts. Moreover, MAC count seemed to mirror SCC patterns, with the worsening of inflammation. Differently, PMN-LYM count exhibited patterns of associations with milk Na and K contents attributable more to LYM than PMN, given the non-pathological condition of the vast majority of the investigated population. An interesting association was observed for milk S content, which increased with increasing of inflammatory conditions (i.e., increased SCC and MAC count) probably attributable to its relationship with milk proteins, especially whey proteins. Moreover, milk Fe content showed positive associations with the PMN-LYM population, highlighting its role in immune regulation during inflammation. Further studies including individuals with clinical condition are needed to have a comprehensive view of milk mineral behavior during udder health impairment.