Effects Of Lactococcus Lactis JCM5805 On The Growth,Intestinal Microbiota And Resistance To Streptococcus Agalactiae Of Nile Tilapia (Oreochromis Niloticus)

Tilapia is one of the most important species of farmed fish of freshwater aquaculture in China.However,in recent years,the high mortality of streptococcal disease has brought serious economic losses to tilapia culture.Probiotics（especially the antagonistic probiotics）can reduce pathogenic bacteria by competitive exclusion,provide nutrients and enzymes to promote host growth,enhance immune response by immune stimulation,and do not cause secondary pollution problems.In view of this,obtaining Streptococcus agalactiae antagonizing probiotics suitable for tilapia culture is of great practical significance for improving the resistance of tilapia and reducing the use of antibiotics.This study was to assess the effects of the potential probiotic strains with special antagonistic activity on Nile tilapia.Comprehensive evaluation of effects on growth,survival,immunity,disease resistance,and intestinal microbiota provides a solid theoretical basis for subsequent commercialization and application of the potential probiotic strains.Main findings are as follows:1.The four initially screened antagonistic strains were applied to Nile tilapia juvenile period.After feeding for 6 weeks,the probiotic supplement groups were feed the basal diet（without probiotics）for another 1 week for intestinal microbial community research.The experiment was carried out in 21 tanks(50L tank^-1,60 fish in each tank).Fish was fed twice daily.The results of this study showed that LR+LL and LL treatments significantly increased weight gain（WG）and feed conversion ratio（FCR）（P<0.05）.Survival rate（SR）of Nile tilapiain LL was significantly higher than CK group（P<0.05）.Gut microvilli length and density and lyzc gene expression were significantly increased in LL group（P<0.05）.Gut microvilli length was significantly higher in LR+LL and BC groups than that in CK（P<0.05）.S.agalactiae resistance of Nile tilapia in LL,BC,LR+LL and BS+BC groups were significantly higher than that in CK（P<0.05）,and the LL group had the highest disease resistance.The results of high-throughput sequencing showed that the main bacteria on phylum level in CK and BS groups was Fusobacteria,and the Proteobacteria in other groups.On genus level,Cetobacterium was the main bacteria in CK and BS groups,but the main bacteria in other groups was Rhizobium,which mainly included Rhizobium radiobacter,and this kind of bacteria can produce coenzyme Q10,that had the effect of improving body immunity.Plesiomonas,which includes potential pathogens of fish,were significantly decreased in the probiotic-fed groups（except for BS）（P<0.05）.A significantly higher presence of JCM5805 in the guts of LL fish during the period of probiotic application.One week post cessation of probiotic administration,the composition of intestinal bacterial community in each test group remained in stable state,which indicated that the cessation of probiotics supplement did not cause disturbance of host intestinal microbes.The above results indicated that JCM5805 is an antagonize probiotic that is suitable for Nile tilapia culture.2.The dosage and frequency of probiotics will affect the application effects of probiotics.This study mainly focused on the effects of different concentrations and application frequency of JCM5805 on the growth,immunity and disease resistance of Nile tilapia.The results indicated that except for T1,the final weight of each group was significantly higher than that of C group（P<0.05）,and the FCR was significantly lower than that of C group on the 4th and 8th weeks（P<0.05）.The specific growth rate of each test group was significant higher than group C on the 4th and 8th weeks（P<0.05）.At the8th week,the levels of skin mucus lysozyme were significantly higher in the test groups than in the C group（P<0.05）,T3 was the highest,followed by T2 and T4.The shin mucus lysozyme level increased after S.agalactia challenge.At the 8th week,except for the T1,the serum peroxidase,alkaline phosphatase,lysozyme,superoxide dismutase and serum complement activities of the experimental groups were significantly higher than the C group before and after challenge（P<0.05）,and it has increased after S.agalactia challenge.At the 8th week,the expressions of TNF-α,IL-1β,hsp70 and TGF-βin the liver,spleen and head kidney of each test group were increased in different degrees except for T1.But overall,the T2,T3,and T4 groups are more effective.At the 8th week,the intestinal lipase,amylase and trypsin were significantly higher in the experimental groups than in C group（P<0.05）.After challenge with S.agalactiae,the survival rate（SR）of each test group except for T1 was significantly higher than that of the C group（P<0.05）,among which the T3 group has the highest SR,which followed by T2 and T4.The results indicated that JCM5805 promoted the growth of Nile tilapia,increased their non-specific immunity,improved intestinal digestive enzyme activity,and improved resistance to S.agalactia.It is most effective when JCM5805 was fed intervally at 1×10⁷cfu g^-1 and1×10⁸cfu g^-1 or continuously fed at 1×10⁶cfu g^-1 in this study.3.This experiment mainly analyzed the effects of JCM5805 on intestinal bacterial colonization and immune regulation of Nile tilapia in the early development stage.The results showed that MyD88 and IRF7 were significantly elevated on days 5 and 10 in the T2 group.TLR9 and IFNαwere also significantly elevated on days 5,10 and 15 during probiotic application of T2（P<0.05）.One month after the cessation of probiotics administration,no significant difference was observed in the expression of these genes（P>0.05）.After JCM5805 was added for 15 days,the expression levels of TNF-α,IFN-γ,hsp70 and IL-1βMrna in T2 intestine were significantly higher that that in C group（P<0.05）.The larvae were fed probiotics for 15 days and were infused with Streptococcus agalactiae strain WC1535 at a final concentration of 1×10⁶cfu ml^-1.Thesurvival rate of T2 was significantly higher than that of the C group（P<0.05）.Microbial characterization by Illumina HiSeq sequencing of 16S rRNA gene amplicons showed the significantly higher presence of JCM5805 in the guts of T2 after 15 days of probiotic continuous application.Although JCM5805 was below the detection level after the cessation of probiotic for 5 days,the gut microbiota of the exposed tilapia larvae in T2 remained clearly diff erent from that of the control treatment after the cessation of probiotic administration.These data indicated that a high concentration of the probiotic strain JCM5805 upregulated the expression of IFNαvia the TLR7/TLR9-Myd88 pathway and enhanced disease resistance of larvae.JCM5805 was only transiently detected and thus was not included in the stable larval microbiota.The early microbial exposure of tilapia larvae aff ects the gut microbiota at later life stages.Preliminary analysis showed that up-regulation of related genes in the signaling pathway is associated with the presence of JCM5805 in the intestine fish.4.In this experiment,the whole genome and secreting antibacterial substancesof JCM5805 were analyzed.The intestinal metabolome were quantitatively and qualitatively analyzed based on the LC-QTOF platform.And together with the combined analysis of intestinal metabolites and bacteria will help to reveal the material basis and mechanism of JCM5805 affecting host growth and immunity.The whole experiment was divided into two groups.The control group（C）was fed with the basic feed,and the experimental group（T）was added JCM5805 at concentration 1×10⁶cfu g^-1.The fish（6.34±0.10g）were reared in tanks（100L）for 8 weeks.The results showed thatfunctional gene annotation of JCM5805 mainly related to carbohydrate metabolism（17%）,general metabolism（13.43%）and amino acid metabolism（9.48%）.And participated in the formation of key enzymes in many metabolic pathways.The active substances produced by JCM5805 existed in fermentation and belonged to secretory antimicrobial substances,which could precipitate under acidic conditions.The antagonistic active compound had good temperature and pH tolerance,and was not sensitive to protease.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.The significantly different metabolic pathways of microbial community function between each group mainly include:carbohydrate metabolism,nucleic acid metabolism,energy metabolism and translation,etc.Principal component analysis of intestinal metabolites showed that the two groups of samples were separated well,and the contribution rates were 52.2%and 24.1%,respectively.The difference multiple,the P value of the Student’s t test and the VIP value of the OPLS-DA model were combined to screen for differential metabolites.A total of 545 significant differential metabolites were obtained,of which447 were up-regulated and 98 were down-regulated.The KEGG annotated results of these differential metabolites show that they are mainly related to metabolism,human disease,organismal systems and environmental information processing.Which mainly included Metabolic pathways,ABC transporters,Cysteine and methionine metabolism,Biosynthesis of amino acids,Arachidonicacid metabolism and Protein digestion and absorption,etc.Within the difference metabolites,histamine,acetyl-L-cysteine and L-cystine were related to the regulation of host immunity and growth.The combined analysis of intestinal metabolites and intestinal flora showed a significant correlation（positive or negative correlation）between the distribution of some intestinal bacteria and the metabolites.Weighted correlation network analysis and coinertia analysis showed that Firmicutes and Proteobacteria were the main causes of the differential distribution of intestinal metabolites.The target strain JCM5805（OTU1）was significantly positively correlated with the distribution of metabolite L-cystine in the intestine（CC>0.90,CCP<0.01）.