| Ictalurus punctatus was introduced into China from the United States in 1984.Due to the characteristics of rapid growth,strong adaptability and rapid popularization,it has become an important breeding species in our country.However,along with the increasing farming scale and breeding density,the problem of disease is becoming more and more serious.Serious infectious diseases had happened in Sichuan,Guangxi,Hunan,Hubei and other places in China,and the drug resistance of pathogenic bacteria becomes more and more prevalent.At present,only 11 kinds of antibiotics in 20 dosage forms are permitted in China,and the number is gradually reducing.If no better approach for controlling the infectious disease is developed,there could be no effective drug to intervene the epidemic.Probiotics was thought to be an effective,environment-friendly alternative to antibiotics,and it was used to control disease and improve water quality in aquaculture.Lactic acid bacteria(LAB)is one of the widely used probiotics,mainly as an additive to feed to improve livestock's healthy.Due to the limitation of methods,the effect of LAB on gastrointestinal microbiota and its colonizing character were still ambiguous.In this paper,colonizing character of Lactobacillus casei(Lc)YYL3 and Lactobacillus plantarum(Lp)YYL5 were investigate in Ictalurus punctatus.In order to clarify the mechanism of Lc-YYL3 and Lp-YYL5,both lactobacillus were added to feed and next generation sequencingwas used to investigate their effects on gastrointestinal microbiota.Based on Picrust functional prediction,the metabolic pathways were analyzed.(1)21 strains of LAB were separated from commercial probiotics,molasses,brownsugar and intestine of Ictalurus punctatus,then identified to 10 species of LAB by 16 S r DNA sequencing,these 10 LAB were Enterococcus faecium,Lactobacillus buchneri,Lactobacillus casei,Lactobacillus plantarum,Lactococcus lactis,Lactobacillus coryniformis,Leuconostoc mesenteroides,Lactobacillus fermentum,Enterococcus avium and Enterococcus hirae.In vitro antagonistic activity,the resistance to bile and adherence assay were determined on these LAB species.The result showed that L.casei YYL3,L.plantarum YYL5,L.plantarum YYL6,L.lactis TM3 and L.fermentum HT3 had higher antagonistic activity on three kinds of pathogenic bacteria(Yersinia ruckeri,Aeromonas hydrophila and Edwardsiella ictalari)on Channel catfish.L.plantarum YYL5,L.plantarum YYL6,L.casei YYL3 and L.lactis TM3 can survive at 0.2% bile.L.casei YYL3,L.plantarum YYL5 and L.fermentum HT3 showed relatively better adhesion to Caco-2 cell,with the adhesion rate above 5%.From the test results,we obtained two kinds of LAB which had higher antagonistic activity of the pathogens,better tolerance and adhesion ability.(2)Bacillus licheniformis(Bli)A1 and Geobacillus stearothermophilus(Gs)were separately used as positive control and negative control for colonizing character experiment of Lc-YYL3 and Lp-YYL5.Fully water exchanging and feed sterilization with Co60 irradiation were employed to avoid their influences on gastrointestinal microbiota of Ictalurus punctatus.A modified p H 5.0 MRS plate with higher selectivity for lactobacillus and a high temperature selective culture method for Bli was assessed for its sensitivity and specificity,and then it was employed to investigate the dynamics character of exogenous Lactobacillus in intestine.The result showed,by exchanging the whole water and feed sterilization,the concentration of Lactobacillus and Bli were reduce to <1cfu/ml in water and were not detected in feed(5.0×102cfu/g LAB and1.4×104cfu/g Bli before irradiation).It took 21 days that Gs inert control was completely excreted from intestine after withdrawing exogenous supplement.Positive control Bli-A1 could sustained at ~1.3×102cfu/g in intestinal content for 28 days at least;while no Lactobacillus was detected from 14 days.In conclusion,Lc-YYL3 and Lp-YYL5 cannot effectively colonize the intestine of Ictalurus punctatus.(3)To investigate the beneficial character of lactobacillus on Ictalurus punctatus,the fishes were fed with diet complemented with 3.0×108 cfu/g Lc-YYL3 and Lp-YYL5 for 4 weeks.Then Growth performance,survival rate,serum non-specific immunity parameter and survival rate after challenge test were determined.Next generation sequencing was used to clarify the influence of Lc-YYL3 and Lp-YYL5 on intestinal microbiota.The result showed that Lc-YYL3 significantly improved growth performance.The terminal body weight(114.67±10.75)g,average body weight gain(47.49±1.62)g and specific growth rate(1.90±0.05)% of Lc-YYL3 group were significantly higher than control and Lp-YYL5 group(P<0.05),while feed conversion rate were significantly lower than CG and Lp-YYL5 group(P<0.05).Serum lysozyme activity of Lc group was significantly higher than CG and Lp group,and there was no significant difference among the groups(Lc,Lp and CG)of SOD activity and alternative complement pathway activity(P>0.05).Lc-YYL3 significantly improved anti-infection ability of Ictalurus punctatus to Edwardsiella ictalari with higher 15-days accumulative survival rate(31.25±6.25%)than control group(CG)(8.33±9.55%)and Lp group(16.67±9.55%)(P<0.05).There was no significant difference between Lp group and CG(P>0.05).Lc-YYL3 and Lp-YYL5 reduced the intestinal microbiota OTUs abundance and diversity.After ceasing both Lactobacillus for 2 weeks,the abundance of Lc-2wk group still lower than CG-2wk(P<0.05),while the OTUs diversity recovered gradually.The OTUs abundance of Lp-2wk recovered,while the diversity was still lower than CG-2wk(P<0.05).Lactobacillus was observed a significantly higher presence in the guts of Lc(85.39%)and Lp(84.58%)fish during the period of probiotic application,but the proportion of Lactobacillus was lower than 0.05% in Lc and Lp groups 2 week post cessation of probiotic administration.The intestinal microbiota of Lc-2wk and Lp-2wk had a significant change after 2 week cessation of probiotic administration.Pseudomonas,Ancylobacter,Neorhizobium,Mesorhizobiumand Brucella were the 5 top dominant bacteria of Lc-2wk group.Whlie Cetobacterium was the dominant bacteria of Lp-2wk group,which gradually recovered to pre-feeding intestinal microbiota.Linear discriminant analysis effect size(LEf Se)demonstrated that there were 39 different taxons between Lc group and CG,and 51 different taxons between Lp and CG.After ceasing probiotics,there were 56 different taxons between Lc-2wk and CG-2wk.Comparing with Lc-2wk,CG-2wk had more abundant taxons on 3 phyla,4 classes,5orders,7 families and 6 genura(Cetobacterium,Romboutsia,Paludibacter,Akkermansia,Plesiomonas and Aeromonas).It suggested that Lc-YYL3 and Lp-YYL5 can reduced the proportion of opportunity pathogen in intestine of Aeromonas.Analysis of predicted functions(PICRUSt)showed that the Lc significantly increased some potential functions of intestinal microbiota involved in lipid,other amino acids,terpenoids and polyketides,xenobiotics biodegradation,carbohydrate and nucleotide,in accordance with metabolic changes observed in terms of weight gain,specific growth rate and specific growth rate.Comparing with Lp,Lc showed more suitable for additives using on Channel catfish,which could improve growth performance,enhance immune status and disease resistance,and affect the gut microbiota of Channel catfish.In conclusion,Lc-YYL3 and Lp-YYL5 cannot effectively colonizing intestine of Ictalurus punctatus.Lc-YYL3 can improve growth performance,non-specific immunity and anti-infection ability to Edwardsiella ictalari.Both Lc-YYL3 and Lp-YYL5 can regulate intestinal microbiota.Metabolism related gene is much higher in Lc-YYL3 group,and it may be an explanation to the better growth performance and immunity. |
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