Ozone micro/nanobubble treatment to prevent the transmission of ecytonucleospora hepatopenaei in pacific white shrimp (Penaeus vannamei)

Abstract

Ecytonucleospora hepatopenaei (EHP) is a significant threat to Pacific white shrimp (Penaeus vannamei) aquaculture, causing severe growth retardation and substantial economic losses. This study aimed to evaluate the application of ozone micro/nanobubbles (OMNBs) for preventing the transmission of EHP in Pacific white shrimp, as well as to assess their effects on the structural integrity of EHP spores. The stability of OMNBs was evaluated by measuring the depletion rate of ozone in culture water. The results indicated that an initial ozone concentration of 2 mg L-1 decreased to a safe level (2 mg L-1) for 24 hours. The treated water was then applied to the treatment group, while the positive control group received untreated contaminated water. The negative control group received only artificial seawater. The experiment was conducted for 15 consecutive days. In addition, the impact of OMNBs on the structural integrity of EHP spores was assessed using scanning electron microscopy (SEM). PCR analysis showed a complete reduction in EHP infection in the treatment group (0%) compared to the positive control group (70%). Quantitative real-time PCR revealed that the EHP DNA copy number in the treatment group ranged from 4.50 x 101 to 7.96 x 102 copies per 50 ng, which are higher than that found in positive control (ranged from 2.91 × 10¹ to 3.29 × 104 copies per 50 ng of hepatopancreas DNA). SEM analysis demonstrated that exposure to OMNBs for 24 hours caused severe structural damage to EHP spores, including membrane rupture and deformation. These findings indicate that ozone nanobubble technology provides a stable and potent oxidative environment for pathogen inactivation, offering a promising biosecurity strategy for the sustainable management of EHP in shrimp aquaculture.