| Bacterial disease,which is one of the main disease types for Trachinotus ovatus in deep-sea cage aquaculture area,has caused great threat and loss for the culture industry in Hainan.In this study,the microbial community composition and dynamics were analyzed by 16S rRNA sequencing based on metagenomics;the pathogen of T.ovatus with rot body symptom was determined through 16S rRNA sequencing and the LD50 and drug sensitivity of QT520 were analyzed through artificial infection and antimicrobial susceptibility test;QT520 genome was sequenced and analyzed using PacBio RS ?;a new method-Real-time loop-mediated isothermal amplification(RT-LAMP)was developed for the detection of V.harveyi.The main results of this research are shown as follows:(1)Through 16S rRNA sequencing method based on metagenomics,the ACE and Shannon indexes of microbial community were 1517 and 3.78,respectively,indicating high richness and diversity of seawater in Houshui Bays,Hainan,2016.(2)The microbial community structure characteristics were analyzed in seawater samples inside and outside of deep-sea cage in March,May,July,September and November in Hainan.Despite there are great difference for richness and diversity of microbial community in Houshui Bay in different months,within the year the results showed the Shannon and ACE indexes in aquaculture area(3.9 and 1564,respecitvely)were higher than in control region(3.6 and 1395,respectively),and the relative abundance of Vibrio inside the cage(8.95%)was higher than outside the cage(2.7%)and control region(1.09%).(3)In seawater samples the peak of relative abundance of Vibrio was in May and September(9.91%and 15.85%,respectively),which was consistent to the peak time of bacterial disease of T.ovatus;and in tissue samples the peak of relative abundance of Vibrio was in May,September and November(34.99%?18.94%and 17.94%,respectively),the peak of relative abundance of Streptococcus was in July(91.44%),indicating Vibrio may be the main pathongen of T.ovatus in May,September and November and Streptococcus was the main pathongen of T.ovatus in July.(4)The NMDS analysis between bacterial community structure and environmental factors demonstrated that temperature,total phosphorus(TP),total nitrogen(TN)and chemical oxygen demand(COD)were important environmental factors affecting the bacterial community structure in deep-sea cage aquaculture area in Houshui Bay,Hainan.(5)One dominant strain QT520 was isolated from liver tissues of T.ovatus with rot body symptom in deep-sea cage aquaculture area in Houshui Bay,2016.Through artificial infection test it was confirmed to be the pathogen causing the disease in T.ovatus.Through morphological,physiological and biochemical characteristics and phylogenetic analysis of the 16S rRNA sequences,the strain was identified as V.harveyi and its LD50 for T.ovatus was 2.5×105 CFU/(mL·g),indicating it was high virulence strain.Analysis results of drug sensitivity showed that it was sensitivity to rifampicin,gentamicin and doxycycline,while resistant to tetracycline,streptomycin and kanamycin.(6)Its genome consists of two circular chromosomes and three plasmids,totaling 6,070,846 bp with a 45%GC content.5701 predicted ORFs,134 tRNAs and 37 rRNAs were observed.Phylogenetic analysis of 12 complete genomes of Vibrio sp.using ANI values and single-copy core genes revealed that QT520 was most closely related to ATCC 33843(392[MAV])and ATCC 43516,indicating that QT520 belongs to V.harveyi.(7)V.harveyi QT520 not only includes common virulence factors,including ACF,IlpA,OmpU,Flagellin,Cya,Hemolysin and MARTX etc,also includes specific virulence factor genes(cyaB,hlyB and rtxA),which were found in plasmid p1,suggesting that the pathogenicity of this strain is plasmid associated.Additionally,more genes encoding types ?,?,?,? and ? secretion systems were detected in the genome of QT520 than strains ATCC 33843(392[MAV])and ATCC 43516,suggesting strain QT520 were provided with the capacity for a highly virulent phenotype.(8)The comparative genomic analysis showed genes associated with type ?secretion systems(T1SS)and type VI secretion systems(T6SS)were detected in QT520,but not in ATCC 33843(392[MAV])and ATCC 43516.The numbers of type?,? and ? secretion systems(T2SS,T3SS,and T4SS)related genes in QT520 was 28,32,and 21,respectively;the numbers of T2SS,T3SS,and T4SS related genes in ATCC 33843(392[MAV])was 7,12,and 4,respectively.The numbers of genes T2SS,T3SS,and T4SS related genes in ATCC 43516 was 7,14,and 4,respectively.Greater numbers of genes associated with the T1SS,T2SS,T3SS,T4SS and T6SS were detected in the genome sequence of QT520 than in strains ATCC 33843(392[MAV])and ATCC 43516,suggesting that QT520 possess more secretory systems than ATCC 33843(392[MAY])and ATCC 43516.(9)According to the principle of LAMP method,Real-time loop-mediated isothermal amplification(RT-LAMP)was developed for the detection of V.harveyi through designing specific primers based on toxR genes of V.harveyi QT520 and adding fluorescent dye SYTO-9 to the reaction system.This method can effectively distinguish the V.harveyi and V.campbellii,and its detection limit was 103 CFU/reaction or 25 CFU/mL.In short,through this research,the microbial diversity in deep-sea cage aquaculture area was studied and the relation between microbial diversity and diseases of T.ovatus was explored in Houshui Bay,Hainan.Through artificial infection test,the pathogen of T.ovatus with rot body symptom was determined and the LD50 and drug sensiti-vity of QT520 were analyzed.Furthermore,QT520 genome was sequenced and analyzed and a new method-Real-time loop-mediated isothermal amplification(RT-LAMP)was developed for the detection of V.harveyi.The results of this study will expand our understanding of the occurrence regularity of bacterial disease,the genome,genetic characteristics,and virulence factors of V harveyi,setting the stage for studies of pathogenesis,diagnostics,and disease prevention and promoting sustainable and healthy development of deep-sea cage fish farming. |
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