A novel lytic bacteriophage, Ecolivirus Myo‑P293 (P293), targeting avian pathogenic Escherichia coli (APEC), was isolated from duck farm sewage in Jiangsu, China. P293 formed clear plaques approximately 2 mm in diameter and displayed the characteristic morphology of Myoviridae family, with an icosahedral head (~ 70 nm) and a contractile tail (~ 100 nm), as observed under transmission electron microscopy. Adsorption assays showed that over 70% of phages adsorbed to host cells within 5 min. One-step growth analysis revealed a latent period of approximately 30 min and a burst size of 244 ± 36 PFU per infected cell. P293 exhibited stability across a pH range of 5–9 and at temperature between 20 and 40 °C, but its infectivity was significantly reduced when exposed to temperature ≥ 60 °C or 60% ethanol. The phage effectively disrupted mature E. coli biofilms, achieving approximately 45% clearance after 24 h of treatment, and also inhibited biofilm formation in a dose-dependent manner. Whole-genome sequencing of P293 revealed an 89.5 kb double-stranded DNA genome encoding 95 open reading frames (ORFs), including modules related to structure, replication, lysis, and host interaction. Approximately 40% of the encoded genes are annotated as hypothetical proteins. Phylogenetic and comparative genomic analyses placed P293 within the unclassified Felixounavirus clade, closely related to Escherichia phage wV8 and Salmonella phage Felix O1, while displaying distinct tail fiber gene signatures associated with host specificity. These findings support the potential of P293 as a candidate for phage-based biocontrol strategies against APEC in poultry production.

Background/Objectives: Targeted delivery of antigens to dendritic cells (DCs) is an effective strategy for enhancing vaccine efficacy. Methods: In this study, dual-targeting fusion proteins (GRFT-VHH54 and GRFT-VHH74) were constructed by fusing Griffithsin (GRFT), an algae-derived lectin with enveloped virus-binding properties, to DC-specific binding nanobodies (VHH54 and VHH74). Vaccines were formulated by combining the inactivated H9N2 avian influenza virus with these fusion proteins, and the potential of the fusion proteins to enhance vaccine-induced immunity in chickens was systematically evaluated. For parallel comparison, control groups included H9N2 avian influenza vaccines containing the inactivated virus alone, the inactivated virus with the immune enhancer CVCVA5, and a commercial H9N2 avian influenza inactivated vaccine. Results: At 4 weeks post-immunization, chickens vaccinated with the inactivated H9N2 virus combined with the GRFT-VHH74 fusion protein (1/2 H9+GRFT-VHH74) exhibited significantly enhanced humoral, mucosal, and cellular immune responses compared to those vaccinated with the inactivated H9N2 virus alone or the commercial H9N2 vaccine (p < 0.05). Additionally, chickens in the 1/2 H9+GRFT-VHH74 group exhibited enhanced resistance to the heterologous H9N2 subtype avian influenza virus, achieving a 90% protection rate, which was higher than that of the other groups. Conclusions: These results indicate that the GRFT-VHH74 fusion protein has significant potential for advancing the development of inactivated vaccines against the H9N2 subtype avian influenza. Furthermore, it provides valuable insights for enhancing the immunogenicity and efficacy of inactivated vaccines targeting other avian influenza subtypes.

Aquaculture sediments are increasingly recognized as important reservoirs of antibiotic resistance genes (ARGs). Although thermophilic fermentation is widely used to reduce ARGs and pathogens in manure, most biosafety assessments stop at the fertilizer product itself, leaving unresolved whether these benefits persist after application to aquaculture sediments. Here, we compared inorganic fertilizer (IF), raw manure (RM), and fermented fertilizer (FF) to test whether fermentation confers sustained biosafety benefits in aquaculture pond sediments. After a 6-month co-culture period, sediment samples were analyzed using shotgun metagenomic sequencing, ARG and mobile genetic element (MGE) profiling, antibiotic residue determination, and network analyses. Long-term fertilization significantly altered sediment physicochemical properties, microbial community composition, and resistome structure. Among the three groups, the RM exhibited the highest total ARG abundance and the greatest number of unique ARG subtypes, with significant enrichment of multidrug resistance genes as well as pathogen-, disease-, and host-associated mobile genetic elements (MGEs). In contrast, the FF group showed the lowest total ARG abundance and fewest unique ARG subtypes, along with suppression of pathogen-associated MGEs, indicating that FF can effectively reduce the risk of ARG dissemination. However, the potential impact of residual antibiotics still warrants attention. Redundancy analysis showed that TC and TN primarily explained bacteriome and resistome variation under RM, whereas pH, EC, AP, and AK were more strongly associated with FF. Co-occurrence analysis further suggested that fertilizer-driven microbial community shifts may regulate ARG persistence and potential cross-ecosystem dissemination. Overall, fermented fertilizer attenuated, but did not eliminate, manure-derived resistance risks in aquaculture sediments. These findings support fermented fertilizer as a safer management option than raw manure and highlight the need for integrated risk assessment combining ARGs, MGEs, microbial hosts, and antibiotic residues.