| Pseudomonas plecoglossicida,a bacterium that causes visceral granulomas disease in large yellow croaker(Larimichthys crocea),has resulted in significant economic losses in the aquaculture industry.Currently,enrofloxacin and doxycycline hydrochloride are the main antibiotics used to treat this disease.However,the overuse of antibiotics can increase the number of drug-resistant strains in the body of large yellow croaker,causing environmental pollution and posing a threat to human health.Therefore,it is urgent to find safe and pollution-free alternatives to antibiotics.Some endogenous probiotics can colonize in the fish body and inhibit the growth of harmful pathogens by producing antibacterial substances,as well as regulate the body’s immunity.Screening for probiotics with antibacterial activity is of great practical significance for large yellow croaker aquaculture.In this study,probiotics that have an antagonistic effect on P.plecoglossicida were isolated and screened from the intestine of large yellow croaker using selective media.The antagonistic strains were identified through physiological and biochemical tests and by constructing a 16S rRNA phylogenetic tree.The antibacterial substances were extracted by ammonium sulfate precipitation,and the physicochemical properties of the antibacterial substances were analyzed.The molecular weight of the antibacterial substance was estimated,and the effects of the antagonistic bacteria on the intestinal structure,serum enzyme activity,and immune function of yellow croakers were further studied through feeding trials.The screening of antagonistic strains against P.plecoglossicida from large yellow croaker is expected to enrich the microbial resources for the biological control of visceral granulomas disease and provide a theoretical basis for the application of antagonistic probiotics in large yellow croaker feed.The research results are as follows:1.A total of 228 strains of bacteria were isolated from the selective medium,and 37 potential probiotic candidate strains were screened by PCR amplification of 16S rRNA gene sequences and homology analysis.Among the 37 candidate strains,19 strains were found to produce protease,11 strains produced lipase,and 10 strains produced amylase.Through the agar diffusion method,three strains with antagonistic effects on P.plecoglossicida were screened,namely P1-17,P2-33,and P3-11.Strains P1-17 and P2-33 had the ability to produce proteases and amylases,while strain P3-11 did not produce any enzymes.2.Based on physiological and biochemical characteristics analysis and the construction of a 16S rRNA phylogenetic tree,strains P1-17 and P2-33 were identified as Bacillus velezensis,while strain P3-11 was identified as Enterococcus faecalis.Antibiotic susceptibility testing showed that strains P1-17 and P2-33 not only exhibited antagonistic effects against P.plecoglossicida,but also against several other aquatic pathogenic bacteria,including Vibrio alginolyticus,V.harveyi,Aeromonas hydrophila,and V.Parahaemolyticus.In contrast,strain P3-11 only showed antagonistic activity against P.plecoglossicida,with no inhibitory effect against other pathogenic bacteria tested.Safety tests demonstrated that all three antagonistic strains were non-hemolytic,susceptible to commonly used antibiotics,had few resistance genes,and were non-pathogenic to large yellow croaker in acute toxicity tests,indicating their excellent biological safety.3.Through subculture,strain P1-17 showed good genetic stability.Antimicrobial substances were extracted from strain P1-17 using ammonium sulfate precipitation method,and the highest antimicrobial activity was observed at 5 0%saturation of ammonium sulfate.SDS-PAGE electrophoresis analysis showed that the molecular weight of the antimicrobial protein was approximately 30 KDa.Single-factor experiments were conducted to optimize four factors of the seed age,inoculation amount,initial pH value,and fermentation broth salinity for fermentation culture.The best antibacterial activity of the antimicrobial substance was observed when the seed age was 18 hours,inoculation amount was 1%,salinity was 1%,and pH was 7.0.The thermal stability of the antimicrobial substance from strain P1-17 was good,and it retained 65.85%of its antibacterial activity after treatment at 100℃ for 30 minutes.It exhibited some tolerance to UV irradiation for 60 minutes and was not sensitive to trypsin,proteinase K,or pepsin,but sensitive to papain.4.Addition of the antagonistic bacterium P1-17 to the feed of large yellow croaker significantly increased intestinal villus height and width(P<0.05),enhanced the activity of immune-related enzymes AKP,ACP,and LZM in the serum,and improved CAT antioxidant capacity.P1-17 could also regulate the expression levels of immune-related genes in tissues such as liver,spleen,and head kidney,and upregulate the expression of IgM,MHC-Ⅰα,MHC-Ⅱβ,and CD4 genes in liver and spleen,as well as MHC-Ⅰα,MHC-Ⅱβ,and CD8 genes in head kidney.These results indicate that B.velezensis P1-17 can relatively improve the growth and immune ability of large yellow croaker. |
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