Bovine rotavirus (BRV) is one of the main pathogens that cause acute diarrhea in calves under one month of age. Passive immunization has been recognized as an effective way to prevent and treat BRV infection. Recent studies have shown that 10% of bovine antibodies possess an ultra-long CDR H3 domain, which has been shown to be the smallest antigen-binding domain. Due to the extremely small size of ultra-long CDR H3 antibodies, the phage display method was utilized to obtain ultra-long CDR H3 antibodies targeting BRV, providing a new approach for the prevention and/or treatment of BRV. Here, we report the preparation of BRV-specific bovine ultra-long CDR H3 antibodies obtained by constructing and screening a phage display library containing approximately 8.55 × 10⁹ individual clones. Through three rounds of bio-panning, we identified 92 candidate clones, of which 79 exhibited specific binding activity in phage ELISAs. The recombinant bovine ultra-long CDR H3 antibodies could specifically bind to BRV in ELISAs and cell immunofluorescence assays. The neutralizing activity was further confirmed through virus neutralization tests. In the calf model experiment, the recombinant bovine ultra-long CDR H3 antibodies could relieve the symptoms of diarrhea, reduce both the amount and duration of virus release, and increase the survival in calves experimentally infected with BRV. Therefore, BRV-specific bovine ultra-long CDR H3 antibodies could serve as an effective agent for the prevention and treatment of BRV infection. At the same time, the development of ultra-long CDR H3 antibodies using phage display screening technology provides a new approach for developing biological agents for the prevention and control of infectious diseases in bovines.

Clostridium perfringens is a major livestock pathogen responsible for histotoxic diseases and intestinal infections, posing serious threats to public health and the poultry and breeding industry. Overreliance on antibiotics has driven the emergence of drug-resistant strains and disruption of gut microbiota homeostasis, underscoring the urgent need for alternative control strategies. In this study, a lytic phage designated vB_CppII_Hohhot was isolated from wastewater samples collected from large-scale dairy farms in Inner Mongolia. The optimal multiplicity of infection was determined to be 0.1. pH stability was assessed. The phage was found to remain stable across pH 3 to 11, but no viable phage was detected at pH 12 (detection limit: <1 PFU/mL). Temperature adaptability was also assessed. Peak titers were achieved between 37°C and 40°C, and the titer was observed to fall below the detection limit at temperatures above 45°C. At a constant temperature of 37 °C, the phage rapidly reached the plateau phase within 40 min, with bacterial counts significantly reduced by 14 h. Whole-genome sequencing revealed that the phage genome consisted of 76 open reading frames, with no detectable antibiotic resistance or virulence genes; an endolysin gene potentially responsible for its lytic activity was also identified. In a prophylactic mouse model, phage administration significantly reduced pathological damage. Collectively, these findings provide a newly isolated candidate agent and a theoretical basis for the prophylactic application of phages against C. perfringens-associated infections.