The Effect Of Ozone Disinfection On Mucus Immunity And Internal And External Flora Of Largemouth Bass And Its Potential Risk

Ozone,as an efficient and environmentally friendly disinfectant,is widely used in water treatment and disease control of aquaculture.Excessive ozone residue not only produces direct toxicity to fish and other aquatic animals,but also destroys the immune defense barrier formed by mucus and microbial community on fish body surface,increasing infection risk.This study focuses on the body surface disinfection effect of ozone as well as the potential risks of its application in large mouth bass（Micropterus salmoides）,mainly including the evaluation of effective disinfection time of ozone at a relatively safe concentration and the changes of oxidative pressure and surface mucus immune-related enzyme activities of the fish after each ozone disinfection,and the analysis of the potential effect of ozone disinfection on diversity and structure of skin,gill and intestinal flora by high-throughput sequencing technology.The main results are as follows:（1）Firstly,it was assessed that the median lethal time（LT50）and the effective disinfection time to breeding water and fish body surface under the relatively safe concentration of ozone（0.05±0.01 mg/L）.The results showed that the LT50 was18.56 h.All the bacteria in the breeding water can be killed by entering ozone for18min,during which the average concentration of ozone is 0.03 mg/L.when the ozone concentration was stable at 0.05±0.01 mg/L,the fish were put into the ozone water,and it was found that the bacteria in the gill and skin mucus were killed after 60 min and 90 min,respectively.（2）Ozone water with a concentration of 0.05 mg/L was used to disinfect the body surface of large mouth bass（90 min/day,3 consecutive days）,and study the effect of each ozone disinfection on the antioxidant defense system and mucus immune-related enzyme activities of the fish.The results showed that ozone treatment significantly increased the contents of reactive oxygen species（ROS）,hydrogen peroxide（H₂O₂）and malondialdehyde（MDA）in the serum（p<0.05）,but the variation trend of these indicators was inconsistent during the three ozone treatments.Ozone treatment had no significant effect on the activity of serum superoxide dismutase（SOD）,glutathione peroxidase（GPx）and glutathione（GSH）,only catalase（CAT）activity increased（p<0.05）,but the total antioxidant capacity（T-AOC）decreased significantly（p<0.05）.Ozone treatment can stimulate the increase of lysozyme activity in the skin mucus and the increase of acid phosphatase（ACP）activity in gill tissues（p<0.05）,but lead to the decrease of ACP activity in skin mucus and alkaline phosphatase（AKP）activity in gill tissues（p<0.05）.（3）The potential effects of ozone disinfection on the flora structure in and out of body in large mouth bass were studied.The experimental fish was divided into initial group（a0）,control group（b15）and treatment group（b15）.The treatment group of bass underwent 3 days of ozone surface disinfection（0.05mg/L,90min/day）,and the control group underwent a similar operation in water without ozone.All experimental fish were kept in the same circulating aquaculture system for another 14d to recover the flora,and samples were taken on days 0 and 15.The results showed that all the experimental fish had skin infection symptoms in the convalescence period,and 2 fish died in the treatment group.The analysis of skin and gill mucus and intestinal flora by high-throughput sequencing showed that the Alpha diversity of skin mucus,gill tissue,foregut,midgut and hindgut flora in the treatment group（b15）and control group（a15）was significantly lower than that in the initial group.However,the results of Beta diversity analysis showed that the structure of bacteria in the treatment group was more similar with that of the control group,differed from that of the initial group.In addition,ozone treatment led to an increase in the Flavobacterium,Nocardia,and Mycoplasma bacteria in large mouth bass,most of which are opportunistic pathogens.Although ozone concentration and exposure time were relatively safe for large mouth bass in this study,oxidative damage and disease resistance were still caused.However,this disadvantage of ozone can also occur in other disinfectants or antibiotics.The results of this study provide important evidence and enlightenment for the safety and potential risks of ozone as a fish disinfectant.