In this study, an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the residue depletion of imidocarb (IMD) in bovine tissues, and the drug withdrawal time of IMD was determined. Twenty-five clinically healthy cattle (body weight 300 kg ± 15 kg) were randomly divided into five groups of five cattle each. The cattle were treated subcutaneously injecting a single dose of a generic IMD formulation, at the recommended dosage of 3.0 mg/kg. The five groups of cattle were slaughtered respectively at 96, 160, 198, 213, and 228 days after IMD administration. Samples from the liver, kidney, muscle, fat, and injection site were collected from each animal. After subtilis proteinase was used to digest the tissue, the content of IMD in the samples was analyzed by UPLC-MS/MS method. In conclusion, the method validation results showed that the method meets the criteria, and the longest withdrawal time of 224 days for the liver can be selected as the conclusive withdrawal time to guarantee consumer safety.

As set in the maximum residue limit regulations of the European Commission, this study aimed to obtain the residual parameters in milk with optimized UPLC-MS/MS conditions and to determine the conclusive drug withdrawal period to ensure food safety. In this research, an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed to study cefquinome sulfate’s residue elimination in milk and to calculate cefquinome’s withdrawal period. Twelve healthy cows free of endometritis were selected for the experiment. Before using the drug, the vaginal orifice and perineum of each cow was disinfected. One dose of intrauterine perfusion was used for each cow, followed by an additional dose after 72 h. Before administration and 12 h, 18 h, 24 h, 36 h, 42 h, 48 h, 60 h, 66 h, 72 h, 84 h, 90 h, and 96 h after the last dose, milk (10 mL) was gathered from each cow’s teat and pooled. For the measurement of cefquinome concentrations in milk, UPLC-MS/MS was performed. A calibration curve was generated using linear regression as follows: Y = 250.86X − 102.29, with a correlation coefficient of 0.9996; the limits of detection and the limits of quantitation were 0.1 μg·kg⁻¹ and 0.2 μg·kg⁻¹, respectively. The average recovery of cefquinome was 88.60 ± 16.33% at 0.2 μg·kg⁻¹, 100.95 ± 2.54% at 10 μg·kg⁻¹, and 97.29 ± 1.77% at 50 μg·kg⁻¹. For 5 consecutive days at the three spiking levels, the intra and inter-day relative standard deviations (RSD) were 1.28%–13.73% and 1.81%–18.44%, respectively; the residual amount of cefquinome was less than the maximum residue limit of 20 μg·kg⁻¹, 36 h after administration; and the residual amount was less than the limit of detection (0.1 μg·kg⁻¹) 48 h after administration. The withdrawal time of cefquinome in cow’s milk was 39.8 h, as calculated using WTM1.4 software. In terms of clinical practical use, the withdrawal period of milk was temporarily set at 48 h after the administration of the cefquinome sulfate uterus injection to cows, in accordance with the recommended dose and course.

Background: Antimicrobial resistance among Gram-positive veterinary pathogens has increased the need for more rational and experimentally grounded strategies to prioritize tylosin derivatives for antibacterial development. This study aimed to develop and externally a calibrated, decision-oriented screening workflow for tylosin-derived antibacterial analog prioritization against Staphylococcus aureus and Streptococcus agalactiae. Methods: Publicly available minimum inhibitory concentration (MIC) data were used to construct organism-specific multilayer perceptron models. Model performance was evaluated using a five-fold out-of-fold framework. Calibrated activity probabilities were then combined with precision-oriented thresholds and similarity-based applicability-domain constraints to define prospective Go/No-Go decisions. To examine external transferability, six synthesized tylosin derivatives (A1–A6) were prospectively predicted and subsequently tested using in vitro MIC assays. Results: Internal validation showed stronger and more stable performance for S. aureus than for S. agalactiae. However, closed-loop external validation revealed distinct organism-specific decision behaviors. For S. aureus, the workflow assigned Go decisions to five compounds and included the only experimentally active analog, A6, but also generated several false Go decisions for inactive analogs. For S. agalactiae, all six compounds were classified as No-Go under the primary decision rule, whereas MIC testing showed that four analogs were experimentally active, indicating conservative under-selection in a low-data extrapolation setting. Conclusions: Calibrated probabilities and applicability-domain analysis can improve the transparency and diagnostic value of antibacterial prioritization, but they do not guarantee robust external activity prediction when training-set coverage, threshold transferability, or chemical-space representation is limited. Overall, this study provides an early-stage, risk-aware closed-loop decision-support framework for tylosin-derived analog prioritization and highlights the need for iterative model updating, probability recalibration, and larger external validation before such workflows are used as high-confidence prospective screening tools in veterinary antibacterial discovery.

Lumpy skin disease virus (LSDV), a capripoxvirus that causes substantial animal-health and economic losses in cattle, remains a major veterinary concern, with few specific antiviral options available. Here, a series of semi-synthetic Tylosin A derivatives were evaluated for anti-LSDV activity using an LSDV-eGFP infection model in Madin–Darby bovine kidney cells. Initial screening identified T3 and T8 as the most active derivatives, reducing infectious virus production by 89.6% and 91.0%, respectively, at 200 ppm. Dose–response analysis based on infectious-virus titration yielded IC₅₀ values of 23.02 ppm (95% CI, 18.48–28.33) for T3 and 19.13 ppm (95% CI, 13.87–25.32) for T8. The corresponding CC₅₀ values were 748.3 and 621.4 ppm, resulting in selectivity indices of 32.50 and 32.48, respectively. Mechanistic studies focusing on T3 showed that antiviral activity was most pronounced when the compound was administered after viral entry and during the early post-infection period. T3 reduced viral genomic DNA accumulation beginning at 8 h post-infection while largely preserving early viral gene transcription and suppressing DNA-replication-dependent intermediate and late gene expression. This phenotype closely resembled that induced by the DNA synthesis inhibitor cytarabine (AraC), supporting an inhibitory effect of T3 at or upstream of the viral DNA replication stage. Proteome-wide molecular docking did not reveal preferential docking of T3 to the selected viral DNA replication-associated proteins, and direct target engagement remains to be established. Collectively, these findings identify T3 and T8 as promising anti-LSDV lead compounds and support further optimization of the Tylosin A scaffold, while mechanistic studies suggest that T3 exerts its antiviral effects primarily during the viral DNA replication phase.

Pasteurella multocida (P. multocida) is not only the core pathogen of bovine respiratory disease (BRDC) but also a significant zoonotic agent, posing a dual threat to global animal husbandry and public health. This study utilized untargeted metabolomics to systematically dissect the metabolic regulatory network of P. multocida in response to tylosin within a One Health framework. The results revealed significant “defense–growth” metabolic reprogramming: activation of amino sugar and nucleotide sugar pathways (e.g., CDP-glucose) indicated cell wall remodeling, while directional shifts in the phenylalanine–tyrosine network directed flux toward defensive secondary metabolites. Concurrently, amino acid disorders and the overactivation of the ABC transporter system exacerbated an internal energy crisis, characterized by a shift from respiration to glycolysis, ATP depletion, and ROS accumulation. SEM observations confirmed membrane integrity disruption and cytoplasmic leakage. Crucially, this metabolic stress and the transition into a “persister-like” dormant state are closely linked to the adaptive expression of antimicrobial resistance (AMR) genes. Under the selective pressure of tylosin, these metabolic perturbations may facilitate the emergence and horizontal transfer of resistance determinants, which can circulate through the animal–human–environment interface. By revealing the metabolic physiological basis of tylosin’s action and its role in inducing bacterial tolerance, this study provides critical theoretical insights for antimicrobial stewardship, aiming to mitigate the risk of AMR transmission and preserve the efficacy of macrolides for both veterinary and human medicine.

Background: Rifaximin is a non-aminoglycoside antibiotic utilized for the treatment of mastitis in cows, but its milk disposition kinetics, residue, and bacteriological status in lactating cow milk have hardly been reported. This study aimed to assess the milk disposition kinetics and residue of rifaximin in milk and to evaluate the bacteriological status in milk after intramammary treatment with rifaximin. Methods: An ultra-high-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) approach was developed to assess rifaximin concentrations in milk. Milk disposition kinetics parameters of rifaximin in cow milk were obtained by non-compartment model analysis. Rifaximin residues in milk were analyzed up to 108 h post-administration to estimate the withdrawal period. Clinically, the efficacy of Rifaximin Intramammary Infusion (Lactating Cow) was evaluated in mastitis cases caused by various pathogens and compared with lincomycin as the control drug, including clinical cure rate, bacteriological cure rate, and somatic cell count (SCC) at D21 post-treatment. Results: The C_max of rifaximin in milk was 54,273.3 ± 12,421.32 ng/mL, the area under the curve (AUC) was 340,731.8 ± 43,968.82 h⋅ng/mL, the T_1/2 was 5.57 ± 0.68 h, the mean resident time (MRT) was 7.3927 ± 1.34 h, and the area under the moment curve (AUMC) was 2,475,745 ± 230,305.1 h⋅h⋅ng/mL. Based on rifaximin residues in milk, the withdrawal period for cow milk was calculated to be 95.1 h. Clinically, Rifaximin Intramammary Infusion (Lactating Cow) demonstrated a clinical cure rate of 83.33% and a bacteriological cure rate of 76.67% in mastitis cases caused by various pathogens, with both rates being 10% higher than those of lincomycin. At D21 post-treatment, the rifaximin group had a significantly lower SCC than the lincomycin group (p < 0.05). Conclusions: Rifaximin exhibits favorable milk disposition kinetics, an acceptable withdrawal period of 95.1 h, and good clinical and bacteriological cure rates in bovine mastitis. These findings support rifaximin as a useful intramammary option and contribute to rational antimicrobial use and milk safety in dairy.