| Tilapia is an excellent species widely farmed in tropical and subtropical freshwater and brackish waters,and it is also one of the greatly popular species in southern China.However,the outbreaks of Streptococcosis due to Streptococcus agalactiae infection have severely limited the healthy development of tilapia in China since 2009.In recent years,infections caused by various pathogens including Edwardsiella tarda,Aeromonas schubertii and Aeromonas hydrophila have caused bacterial diseases in tilapia,which increased the difficulty of prevention and control of bacterial diseases in tilapia.Nowadays,the prevention and control of tilapia streptococcosis depend on antibiotics and other drugs,but the abuse and long-term use of antibiotics will lead to drug resistance of pathogens and drug residues in aquatic products.Therefore,ecological control is particularly important,and it is of great practical significance to seek efficient and pollution-free alternatives to antibiotics.In this study,a strain was screened from the intestinal tract of Nile tilapia(Oreochromis niloticus),which has strong antagonistic effect on common fish pathogenic bacteria,and the identification and biological characteristics were analyzed.Furthermore,the effects of dietary supplementation on growth,intestinal flora composition,immune function and disease resistance of tilapia were studied in order to enrich the biocontrol microbial resources of Streptococcosis in tilapia,and provide theoretical basis for the application of antagonistic probiotics in tilapia.The main results are as follows:1.Selection and biological characteristics of intestinal antagonistic Bacillus strain of tilapiaA strain named LF01,which antagonizes S.agalactiae,isolated from the intestinal tract of healthy Nile tilapia(Oreochromis niloticus)in this study.The strain LF01 was identified by physiological,biochemical characteristics and molecular biological analysis.In addition,some characteristics and biosafety of the strain LF01 were also determined and analyzed.The results showed the strain LF01 was identified as Bacillus velezensis.The optimum growth temperature,pH,and salinities of the strain LF01 were30℃,7,and 5‰,respectively,and it exhibited the ability to hydrolyze starch and casein.The strain LF01 was sensitive to most antibiotics and resistant only to bacitracin,and showed antimicrobial activity against a broad range of fish pathogens including S.agalactiae,Streptococcus iniae,E.tarda,Edwardsiella ictaluri,Aeromonas hydrophila,A.schubertii,Aeromonas veronii,Aeromonas jandaei and Nocardia seriolae.Biosafety tests suggested that the strain LF01 was not pathogenic to Nile tilapia,Danio rerio,and Channa argus.It was found that B.velezensis LF01 with great biosafety could antagonize several fish pathogens and has the potential to prevent and control a variety of aquatic animal diseases,and has broad application prospects.2.Isolation and characterization of active compounds produced by Bacillus velezensis LF01To investigate the characteristics of the active compounds produced by B.velezensis LF01,the antagonistic substances were obtained by concentrated hydrochloric acid precipitation under the optimum antimicrobial conditions,and its antagonistic properties were tested.At the same time,the stability of the antagonistic substances was conducted.The results showed that the active substances produced by B.velezensis LF01 existed in fermentation and belong to secretory antimicrobial substances,which could precipitated under acidic conditions.The results indicated that the production of antimicrobial substances was one of the reasons for the biocontrol effect.The crude extracts showed high inhibitory effect at-20℃and-80℃,remained activity even at 121℃.The antagonistic effects of the crude extracts were stable at a pH of 4.0 to 10.0 and remained activity even at pH 2.0 or pH 12.0.No significant differences in antibacterial activities of the active compounds via proteinase were observed in the treatments.Briefly,the antagonist was well tolerated to temperature,acid-base and was not sensitive to the proteinase,thereby confirming that it was a non-protein.Moreover,the time-kill showed that the number of viable cells of S.agalactiae in the active compounds declined sharply in a short time,even disappeared,showing strong antibacterial and bactericidal effects.This study indicated that the strain LF01 might have the potential to be used as an antagonistic probiotic,and laid a foundation for further study on the application of antagonistic probiotic LF01 in tilapia feed.3.The effects of Bacillus velezensis LF01 on the growth,intestinal microflora and immune characteristics of the Nile tilapiaThe potential application of B.velezensis LF01 in juvenile Nile tilapia was evaluated by basic experiments and molecular biological analysis.The experiments were fed with different diets:control group with non-supplemented diets,interval group and continuous group with B.velezensis LF01 supplemented diets at 1.0×10~9 CFU/g over a period of 9 weeks.Three weeks as a time point.A feeding experiment was carried to evaluate the effects of dietary administration of probiotics with B.velezensis LF01 on the growth,immune responses,intestinal microflora of the juvenile Nile tilapia and disease resistance to S.agalactiae infection.The results suggested that the growth performances in continuous group were significantly higher than those in CK after 9 weeks of feeding,though no significant differences were observed in those between in interval and continuous groups for the same period.Additionally,there was no significant difference in the survival rate between control and the treatment groups.This exhibited significant enhancement in growth performance compared to CK.And both interval and continuous groups also resulted in the tilapia having significantly higher levels of LZY and SOD activities and no significant difference among TP,ALB,and GLO compared to CK.Meanwhile,the relative percent survival of tilapia fed with B.velezensis LF01 was significantly improved compared to CK,with tilapia fed with probiotics having the highest relative percent survival after continuous feeding 9 weeks.Moreover,among them,three genes in three tissues showed higher expression after feeding compared to CK group.In addition,Fusobacteria,Proteobacteria,and Planctomycetes were dominant in the intestines of tilapia regardless of the diet.Furthermore,the proportion of Edwardsiella in the intestines of tilapia in the interval group(0.01%)and continuous group(0.22%)was significantly lower than that of tilapia fed with the con diet(2.33%).And the number of Plesiomonas in the intestinal tracts of tilapia in the continuous group(0.70%)was also significantly lower than in CK(2.69%)and interval group(2.74%),although no significant difference was detected between the CK and interval group.In summary,the above results clearly indicated that dietary B.velezensis LF01 could have positive benefits for tilapia aquaculture and it has the potential to be developed as a probiotic agent in aquaculture. |
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