The overuse of antibiotics in animal husbandry has driven the search for alternative strategies to combat zoonotic pathogens. Foodborne zoonotic diseases caused by pathogenic bacteria pose a significant threat to human health, and therefore food safety should be a priority. This study investigates the in vitro inhibitory effects of chicken egg yolk immunoglobulin Y (IgY) on the growth and viability of three major foodborne pathogens: Campylobacter jejuni, Salmonella spp., and Escherichia coli. IgY was isolated from immunized hen egg yolks using a modified water dilution method, and its antigen-specificity confirmed through agglutination assays. Growth inhibition was evaluated across multiple doses and time points, revealing a dose-dependent bacteriostatic effect against all tested pathogens. A single dose of IgY (0.5 mg/mL) significantly reduced C. jejuni counts by up to 7 log, while repeated doses were required for Salmonella spp. and E. coli. These findings highlight egg yolk immunoglobulin’s potential as a source of sustainable, effective, ethical, readily available, and inexpensive antibiotic substitutes in livestock management. Future research will focus on validating these results in vivo and exploring large-scale production of IgY for practical application in animal healthcare.

Bovine mastitis causes severe economic losses in the dairy industry and relies heavily on antimicrobial treatments, accelerating the emergence of antibiotic resistance. Avian egg yolk antibodies (IgY) offer a promising, sustainable alternative. This study evaluated the in vitro efficacy of pathogen-specific IgY against major mastitis-causing bacteria isolated from over 100 environmental dairy samples. Laying hens were immunized with inactivated native strains to produce targeted IgY. The extracted antibodies underwent rigorous quality control, including purity, agglutination titers, and endotoxin level assessments. Their inhibitory effects were evaluated in both standard broth medium and a host–pathogen challenge model utilizing bovine teat and lactiferous sinus epithelial cells. The isolated IgY antibodies exhibited high purity and strong antigen-specific agglutination. Functionally, the specific IgY effectively inhibited the growth of targeted pathogens (including Staphylococcus aureus, Escherichia coli, Klebsiella spp. and Streptococcus spp.) in the liquid medium. Crucially, this significant inhibitory effect was consistently maintained in the host–pathogen challenge model, indicating robust pathogen neutralization capabilities. These in vitro findings demonstrate that targeted IgY antibodies can effectively suppress mastitis-related bacteria in environments mimicking the host epithelium. This highlights the potential of IgY technology as a safe, non-antibiotic intervention strategy for bovine mastitis, strongly supporting the One Health approach.