Studies On The Molecular Epidemilogical Characteristics And Analysis Of The Virulence Strain Genome Of Vibrio Harveyi Isolated From Mariculture Area Of South China

Vibrio harveyi is a common pathogen in the coastal areas of south China and has caused serious damage to different types of mariculture animals.In order to study the molecular epidemiological characteristics,explore the pathogenesis,analysis the distribution of virulence genes of V.harveyi isolated from south China,and to develop a rapid detection method,study the antimicrobial resistance,explore the genomic characteristics of V.harveyi pathogen strains,the experiments of the molecular typing,the artificial infection of V.harveyi and the specificity detection biomarker screening,the antimicrobial resistance test,the whole genome sequence analysis of V.harveyi pathogen strains were carried in this paper,the results were as follows:1.A collection of 46 V.harveyi isolated from grow-out culture ponds/cages of south China were typed by RAPD and ERIC-PCR methods,a major class of strains with the similar genetic background were obtained by the two methods.Of which,18 strains clustered together by the ERIC-PCR method were named the ERIC-1 profile.The eighteen strains were determined to be pathogenic by artificial infection with healthy E.coioides at CFU of 107 ml-1 bacteria liquid.The mortality was showed within 2?6 h after injection.It was shorter than existing reports.It indicated that the pathogenic V.harveyi strains in south China had similar genetic background,they might be the typical pathogen in the region.2.The more pathogenic V.harveyi strain GDH11385 was used to infection a variety of aquaculture animals and mice.The results showed that the strain was toxic to E.fuscoguttatus ?×E.lanceolatus?,Trachinotus ovatus,Siganus guttatus,Penaeus vanname,Trichogaster trichopterus,T.microlepis and mice.It indicated that the strain GDH11385 could infect multiple hosts.The LD50 values of GDH11385 for grouper and mice were 8.7×103 CFU/g and 9.9×105CFU/g body weight,respectively.The bacterial suspension and extracellular products of GDH 11385 strain were used to infect E.coioides,the results showed that the bacterial suspension could cause the animals to die rapidly,but the extracellular products were.not pathogenic,indicating that the pathogenic factors of GDH 11385 should belong to the bacteria cells.Histopathological examination showed that the bacteria could penetrate into the liver,spleen and brain and other tissues,and caused serious pathological damage to these tissues and organs.Combined with the symptoms of disease and the anatomical results of diseased fish,it inferred that colonization ability of the bacteria was strong.It could quickly colonize the host tissue and organs,and chelate the iron ions of the organization,so that the ability of hemoglobin to transport oxygen of the infected animals greatly reduced,resulting in infected animals death by hypoxia.3.All the 46 strains were screened for the presence of virulence genes that are typical for these bacteria and those found in human pathogenic vibrios such as V cholerae,V parahaemolyticus and V.vulnificus and aquatic pathogenic V.anguillarum.The virulence genes were amplified by PCR with specific primers.The five virulence genes luxR,toxRvh,chiA,serine protease and vhh,typical of Harveyi clade were detected in all the pathogenic isolates.The non-pathogenic strains only contained 1-4 these genes.It indicated that these five genes might be the main virulence genes of this ERIC-1 profile strains.4.According to the ERIC-PCR fingerprints common bands of ERIC-1 pathogenic strains,the specific molecular biomarkers of this class of strains were screened,then the rapid detection primers of these strains were designed based on the biomarkers.The species-specific primers were with high specificity and high sensitivity.It demonstrated that the bacteria,which have important influence in mariculture in south China,could be specifically detected by using ERIC-PCR fingerprint-based amplification.5.Five pathogenic V.harveyi strains were isolated from grouper hatcheries in the main grouper breeding base Hainan province.Genotypic assay of these 5 strains were conducted by enterobacterial repetitive intergenic consensus(ERIC)-PCR,the results showed that all the 5 strains had the similar genotype profile,which was as the same as the ERIC-1 profile.The resistance of all the 23 ERIC-1 profile strains(18 from grow-out culture ponds/cages,5 from grouper hatcheries)to ten categories,26 different antibiotics was investigated.The results showed that in grow-out culture ponds/cages,the strains were susceptible to chloramphenicols,quinolones,sulfonamides,and were resistant to ?-lactams,aminoglycosides,tetracyclines,while in grouper hatcheries,the strains were resietant to all the ten categories antibiotics more or less.Therefore,our study indicates that bacterial multiple antibiotic resistance(MAR)is more widespread in grouper hatcheries than ponds/cages.6.The high quality of genomic DNA of V.harveyi strain GDH11385 was obtained by removing the polysaccharide.The purified DNA was subjected to high throughput sequencing using the third generation sequencing platform,the Pacbio RSII system.Then the sequences were spliced and assembled,and two main chromosomes and three plasmid sequences were obtained.Genomic annotation,gene functional classification,bacterial key virulence gene analysis and comparative genomics analysis were carried out to provide a theoretical basis for the control of Vibriosis on aquaculture.