Na Yu

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.

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.

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.