Study On The Disease Of Nile Tilapia Oreochromis Niloticus Infected With Aeromonas Schubertii And Its Prevention Of Probiotics

Aeromonas is not only an important zoonotic bacteria that causes severe disease in infected mammals and humans,but also the main pathogen of aquatic animals.Aeromonas diseases severely restricts the healthy development of the aquaculture industry.Antibiotics are more and more restricted in fish disease control due to its drug resistance and drug residues problems.Probiotics have become the most promising alternatives.In this paper,the pathogenic bacteria are identified by the method of pathogen isolation;then the impact of the pathogenic bacteria on the host immune indicators and immune-related genes is studied;at the same time,in order to prevent disease outbreaks,probiotics screening and its effects were carry out of and effect research.The main research contents and results are as follows:1.Isolation and identification of Aeromonas schubertiiThere were outbreaks of tilapia diseases in brackish water lakes in Haikou City,Hainan Province from September to October 2017.To determine the cause of this outbreak,four dominant strains（ASL-1,ASB-2,ASB-3 and ASS-4）were isolated from the diseased tilapia.The challenge test showed that the ASS-4 was the most virulent and the 7-day LD₅₀was 4.91×10⁴ CFU/g.Physiological biochemical tests and molecular identification（16S rDNA,ela,dnaJ and gyrB）showed that all four dominant strains were A.schubertii.The different infection routes tests showed that the order of cumulative mortality rates of different infection routes,from high to low,was intraperitoneal injection>intramuscular injection>oral>immersion.2.Effect of A.schubertii on tilapia immune indexes and immune-related genesTilapia were infected with A.schubertii（ASS-4）by intraperitoneal injection,and the immune indicators and immune-related genes expression at different tiome points（6 h,12h,24 h,48 h,72 h and 168 h）after infection.The results showed that the ASS-4 stimulated the tilapia’s antioxidant activity（SOD,GSH-PX and MAD）and non-specific immune activity（RB,ACP,AKP and LZM）at an early stage.RT-qPCR detected that the transcription of immune-related genes（COX-2,TNF-α,IL-1β,TGF-β,IL-10 and HSP70）were activated in the tilapia intestine and head kidney tissues.3.Screening and identification of probiotics from tilapia gut and evaluation of their probiotic characteristicsIn order to screen potential probiotics against A.schubertii,414 strains from tilapia gut were isolated using LB and MRS agar medium.By testing hemolytic activity,extracellular protease activity,extracellular amylase activity and antagonistic A.schubertii activity,four potential probiotics were screened out.Based on artificial gastrointestinal fluid tolerance and bio-surface coating formation ability,G45 was selected as a potential probiotic for subsequent experiments.After physiological biochemical and 16S rDNA identification,G45 was identified as Bacillus velezensis.4.Effects of B.velezensis on tilapiaIn order to study the probiotic effect of B.velezensis（G45）on tilapia,G45 and Lactobacillus plantarum（1036 and 58）were continuously fed to tilapia for 10 weeks.The results show that G45 significantly improved the growth performance,antioxidant activity and non-specific immune activity of tilapia.The results of immune-related genes showed that G45 increased the relative expression of COX-2,TGF-β,IL-10 and HSP70,and reduced the relative expression of TNF-αand IL-1βin tilapia.The A.schubertii infection test showed that cumulative mortality rates in G45 group were significantly lower than in the control group.