Abstract
Background:
Listeria monocytogenes is a serious foodborne pathogen affecting public health and animal production. In rabbits, it causes systemic infections, leading to economic and welfare concerns. Rising antibiotic resistance highlights the need for natural alternatives to conventional antimicrobials.

Aim:
This study evaluated the antibacterial efficacy of propolis-pollen nanoemulsions (PP/NE) against L monocytogenes infection in rabbits, comparing its effect with that of ciprofloxacin and assessing its ability to modulate bacterial virulence gene expression.

Methods:
A total of 120 rabbits (10 weeks old) were randomly assigned to six groups (n = 20/group). Group 1 served as a negative control. Group 2 was orally infected with L monocytogenes (1.3 × 10⁹ CFU/mL) for three days. Group 3 received only PP/NE. Group 4 was infected and treated with PP/NE. Group 5 received prophylactic PP/NE before infection. Group 6 received infected ciprofloxacin (30 mL/kg). Bacterial loads in liver and brain tissue samples were measured, and expression levels of the hlyA, inlA, and actA genes were evaluated using RT-PCR.

Results:
Treatment with PP/NE, either alone or combined with ciprofloxacin, significantly reduced bacterial counts compared with the positive controls. The liver and brain counts in the PP/NE+ciprofloxacin group were the lowest (5.67 CFU and 0.00 CFU, respectively). Virulence gene expression was significantly downregulated by day 21, particularly in the combination group, with hlyA, inlA, and actA levels of 0.097, 0.11, and 0.046, respectively.

Conclusion:
PP/NE exhibited strong antibacterial activity against L monocytogenes and significantly reduced the expression of key virulence genes. It has potential as a natural therapeutic or prophylactic agent in rabbit farming and as a food safety enhancer, potentially reducing antibiotic reliance.

Key words: Listeria monocytogenes, Toxin genes, Propolis-pollen nano-emulsion, Antibiotics, Rabbits