This study investigates the effects of quercetagetin (QG) supplementation on the antioxidant capacity, liver mitochondrial function, and cecal microbiota of broilers raised under high-density conditions. A 2 × 2 factorial design with 144 one-day-old WOD168 broilers, which were allocated to two stocking densities (LD: 4 birds per cage, equivalent to 11.1 birds per square meter; HD: eight birds per cage, equivalent to 22.2 birds per square meter) and two levels of dietary supplementation of QG (0 and 20 mg/kg). At the conclusion of day 21, broilers of similar body weights were randomly allocated into four groups (22 to 42 d): control (CON), QG treatment (QG), high stocking density (HSD), and high stocking density with QG supplementation (H_QG). The results demonstrated that HD groups significantly reduced broiler growth performance, including body weight (BW), average daily gain (ADG), and average daily feed intake (ADFI) (p < 0.05). Additionally, HD groups increased serum stress hormone levels (CORT and ACTH), pro-inflammatory cytokines (IL-1β and IL-6) (p < 0.05), while decreasing liver antioxidant enzyme activities (GSH-Px, T-SOD), serum CAT and T-SOD activities, and mitochondrial function (GSH, complex I-III, ATP contents) (p < 0.05). However, dietary supplementation with 20 mg/kg QG significantly alleviated the negative effects induced by HSD, restoring growth performance, stress hormone levels, immune parameters, and liver antioxidant and mitochondrial function. Moreover, QG supplementation markedly improved cecal microbiota composition, enhancing gut health. Correlation analysis revealed a strong association between microbial composition and overall broiler health, indicating that gut microbiota plays a critical role in mediating these beneficial effects. In conclusion, QG exhibits protective effects against oxidative stress, mitochondrial dysfunction, and gut microbiota imbalance induced by high-density rearing, suggesting its potential as a functional feed additive to improve broiler health under intensive farming conditions.

Multidrug-resistant (MDR) Salmonella enteritidis infections cause high mortality and devastating economic losses in poultry, pose severe threats to animal health and food safety, and create an urgent demand for effective antibiotic alternatives. Herein, we developed a cationic liposome-encapsulated bacteriophage endolysin Lys40 (designated Lys40-Lip), and systematically evaluated its therapeutic efficacy in a chick model challenged with Salmonella enteritidis strain S4. Recombinant Lys40 was encapsulated into cationic liposomes with an encapsulation efficiency (EE) of 34.83%. The resulting Lys40-Lip nanoparticles had a hydrodynamic diameter of 137.3 ± 4.1 nm, a high positive zeta potential of +42.5 ± 0.3 mV, and excellent stability, retaining 78.52% of its initial bactericidal activity after 56 days of storage at 4 °C. Following a three-day oral treatment in Salmonella enteritidis S4-infected chicks, Lys40-Lip significantly increased survival rates in a dose-dependent manner (72.22% to 88.89% for low-to-high dose vs. 44.44% in infected controls, p < 0.05) and reduced ileal Salmonella enteritidis S4 colonization by 28.8% compared to free Lys40. Histopathology revealed Lys40-Lip restored duodenal villus integrity and reduced jejunal and ileal inflammation. Serum cytokine analysis confirmed that Lys40-Lip effectively regulated the host inflammatory response, significantly downregulating the pro-inflammatory cytokines IL-1β and IL-6, and upregulating the anti-inflammatory cytokine IL-10. Crucially, liposomal encapsulation overcame the outer membrane barrier of Gram-negative bacteria via charge-driven fusion mediated by its high positive surface potential (+42.5 ± 0.3 mV), enabling targeted delivery of Lys40 without the need for EDTA or other outer membrane permeabilizers. Lys40-Lip significantly improved the therapeutic outcomes of avian salmonellosis via synergistic direct bactericidal activity, intestinal barrier protection and inflammatory response regulation, offering a promising nanotherapeutic strategy for the control of this disease in veterinary practice.

Background: Extraintestinal pathogenic Escherichia coli (ExPEC) poses significant health risks to poultry and humans, with biofilm formation often complicating treatment by enhancing bacterial persistence and resistance. Understanding the genetic mechanisms underlying this lifestyle transition is crucial for controlling infections. This study aimed to investigate the effect of biofilm formation on the transcriptional expression of specific biofilm- and virulence-associated genes in chicken-derived ExPEC strains. Methods: Biofilm formation conditions for three strong biofilm-producing chicken-derived ExPEC strains were optimized using an orthogonal experimental design (L₉(3³)), evaluating culture medium, incubation time, and initial inoculum concentration. Biofilm biomass was quantified via crystal violet staining. Subsequently, the transcription levels of 10 biofilm-associated genes and 17 virulence-associated genes were compared between planktonic and biofilm states using Reverse Transcription-quantitative PCR (RT-qPCR). Results: Optimal culture conditions varied among strains, though nutrient-rich media consistently promoted rapid biofilm formation. Transcriptional analysis revealed significant reprogramming in the biofilm state. Among biofilm-associated genes, flhC, tolA, qseC, mhpB, and bdcR were consistently and significantly upregulated across all strains (p < 0.05). Regarding virulence determinants, the expression of eaeA, LT, fimH, ompF, and iss was significantly upregulated (p < 0.05), whereas Sta levels were significantly reduced (p < 0.05). Conclusions: Biofilm formation induces a distinct transcriptional shift in chicken-derived ExPEC, simultaneously enhancing the expression of key genes involved in biofilm maintenance and pathogenicity. The conserved upregulation of flhC, tolA, qseC, mhpB, and bdcR suggests these genes are critical drivers of biofilm development. Consequently, they represent potential targets for novel therapeutic strategies aimed at preventing E. coli infections and eradicating biofilms in clinical and agricultural settings.

Phage holin Hol41 is a membrane protein with broad-spectrum antibacterial potential; however, its high hydrophobicity and prokaryotic expression toxicity s