Simple SummaryInfectious mastitis causes significant economic losses and affects dairy cattle health and welfare. The present research aimed to test an intramammary-infused therapeutic compound to promote sustained mastitis treatment and improve cure rates in dairy cattle. The effects of intramammary infusion of micronised purified flavonoid fraction (MPFF) were studied as a natural alternative to conventional synthetic antimicrobials and against antimicrobial resistance in mastitis-diagnosed dairy cows infected byStaphylococcusspp. in the late lactation.AbstractInfectious mastitis is the most prevalent health problem in dairy cattle that can result in permanent economic losses on dairy farms. The micronised purified flavonoid fraction (MPFF) is a biocompatible active polyphenolic compound derived from flavonoid glycosides which exhibits several antimicrobial, anti-inflammatory, and phlebotonic properties. The goal was to assess the effects of an alternative therapy for mastitis based on MPFF intramammary infusions in late lactation in dairy cows naturally infected byStaphylococcusspp. The California Mastitis Test (CMT scores) was performed to detect mastitis-positive quarters in twelve dairy farms. All cows were screened for immune response by measuring somatic cell counts (SCCs; cells/mL) in milk samples from each quarter. In addition, bacteriological identification, pathogenic bacterial isolates, and total bacterial counts (TBCs; CFU/mL) were assessed before (day 0, last milking day) and after (day 3 post-calving) MPFF application. Antimicrobial sensitivity patterns of the pathogenic isolated bacteria were evaluated. Finally, cure rates (%) were determined for each MPFF treatment. Around 15 mastitis-related genera were isolated.Staphylococcus aureus(25.2%) and coagulase-negative Staphylococci (CNS; 22.4%) were the most prevalent pathogens. No statistical differences were observed in SCCs and TBCs after low, medium, and high MPFF dose administration inS. aureus-positive mastitis cases (p> 0.05). However, differences were observed in SCCs and TBCs after medium and high MPFF dose administration in CNS-positive quarters (p< 0.05). The pathogenic bacteria isolate reduction after MPFF applications showed a dose-response fashion (p< 0.01) while isolates obtained from controls and low MPFF-treated quarters remained similar, irrespective of the pathogen (p> 0.05). Sensitivity patterns were variable, althoughS. aureusremained resistant, irrespective of the MPFF dose. However, CNS showed a dose-response sensitivity pattern. Finally, the cure rate (%) on day 3 post-partum improved significantly using medium and higher MPFF doses in CNS-positive quarters (p< 0.05). In conclusion, MPFF treatment was found to be more effective for CNS-positive cases in the late lactation due to noticeable dose-specific responses regarding somatic cells, bacterial counts, sensitivity patterns, and cure rates in dairy cattle.