A. hydrophila causes haemorrhagic septicaemia in freshwater fish, with outbreaks leading to up to 80% of losses in some stocks. Previous reports have demonstrated that dietary probiotics can enhance the disease resistance of C. carpio against various pathogens, including A. hydrophila. This study isolated carp autochthonous candidate probiotic strains, with in vitro antagonism against a range of fish pathogens, and used aqueous administration to investigate the potential to inhibit A. hydrophila in the rearing medium and on the mucosal surfaces of mirror carp.
Intestinal mucosa and digesta samples from C. carpio (n = 5) were homogenized in phosphate-buffered saline (PBS). Homogenates were cultured on tryptic soy agar (TSA) plates and isolated colonies were tested in vitro for antagonistic activity against A. hydrophila, Streptococcus iniae, Vibrio anguillarum, Vibrio parahaemolyticus, Yersinia ruckeri, and Pseudomonas anguilliseptica. Out of 150 isolates, 16 displayed antagonism against A. hydrophila. Of these 16, 87.5% were antagonistic against S. iniae, 50% were antagonistic against V. anguillarum, 6.3% were antagonistic against V. parahaemolyticus, 12.5% were antagonistic against Yersinia ruckeri, and 87.5% were antagonistic against Pseudomonas anguilliseptica. Two promising isolates (C24 and C72) were used in pathogen-mucosal colonisation studies. In order to reduce negative impacts on welfare by challenging live fish, exposure assays were conducted using whole fish bodies immediately post-mortem. C. carpio (mean weight, 18.0±3.8 g) were euthanized and distributed randomly into four exposure treatments (n = 5): (T1) control, exposed to sterile PBS, (T2) PBS containing A. hydrophila, (T3) PBS containing A. hydrophila and probiotic candidate C24, and (T4) PBS containing A. hydrophila and probiotic candidate C72. After 90 min exposure, the fish skin, gill, fins, and rearing medium were sampled, and homogenates were spread onto Aeromonas agar plates (incubated at 25°C for 24-36 h) to determine Aeromonas levels. A. hydrophila was not detected in the rearing medium or mucosal tissues in the control group after the exposure. The levels of A. hydrophila in treatments T2 and T3 increased by 82% and 89%, respectively, demonstrating the ability of A. hydrophila to proliferate in the PBS solutions exposed to carp mucus and the inability of isolate C24 to inhibit A. hydrophila proliferation under such conditions. However, candidate C74 significantly (P < 0.05) retarded the growth of A. hydrophila in treatment T4 (with A. hydrophila levels only increasing by 34%, vs 82% in the control). The effects on A. hydrophila mucosal colonisation levels were also investigated (data not shown). Further studies are being conducted to observe the effects on mucosal colonization levels on a range of other aquacultured freshwater fish species.