Revitalizing pond culture system: Harnessing the power of composite nanopeptide C[sbnd]I20, 1,3-1,6-β-glucan, and anthocyanidin biotherapy as an antibiotic substitute for efficient management of bacterial diseases in bullfrogs

Abstract

Bacterial diseases in aquaculture ponds have significant negative effects on sustainable development. Antibiotic resistance in aquatic pathogens has reduced the effectiveness of traditional treatments. To address this, composite additives that boost the natural defense system are being explored. In this study, we focused on four common symptoms in bullfrog ponds: amyosthenia, black body color, crooked head, and pythiosis. We also observed overlapping symptoms such as amyosthenia + crooked head and black body color + pythiosis. The overall incidence rate of these symptoms was 85.6%, with Aeromonas hydrophila and Elizabethkingia miricola identified as the responsible pathogens. To treat bullfrog bacterial diseases, we used two treatments: florfenicol (FF) at 20 mg/Kg and a composite I20 (CI) containing nanopeptide CI20 (2 g/Kg), β-glucan (1 g/Kg), and anthocyanidin (1 g/Kg). We implemented a staged therapy approach, comprising initial assessment, self-cure intervention, and continuous treatment stages, along with continuous monitoring to assess the effectiveness and usage protocol of CI. In the initial therapy stage, oral CI or florfenicol improved body color and rapidly reduced morbidity rate and daily mortality rate. CI biological agent exerted anti-bacterial infection by improving mucosal barrier, antioxidant enzyme activity (T-AOC and TSOD), and innate immunity index (LZM activity, leukocyte number). The CI treatment demonstrated significant reductions in tissue injury (intestine, liver, spleen) and tissue bacterial load. Following the stage therapy, the CI group exhibited lower morbidity and daily mortality rates compared to the FF group. Additionally, the total protection rate of the CI group surpassed that of the FF and control groups during the stage therapy. The antibiotic resistance and drug resistance genes (FIoR and FexA) and virulence genes (HxlR, ClpP, OmpA, and FlaA) in A. hydrophila and E. mirabilis were markedly induced by florfenicol treatment, respectively. In continuing therapy stage (42 days), the morbidity, protection rate, and daily mortality of the CI group were better than that of the FF group. In conclusion, composite I20 biotherapy effectively treats the bacterial disease of bullfrogs in pond system, providing a prospectively novel strategy for controlling bacterial disease in aquaculture.