| Vibrio harveyi is an important opportunistic pathogen,which can infect a variety of marine fish and invertebrates.In recent years,with the expansion of my country’s marine aquaculture industry,the impact of Vibrio harveyi on aquaculture animals has been increasing,and its host range has also expanded.Currently,there are many commercial vaccines against V.harveyi,but the immune effect is different when used in different regions,which may be due to the antigenic difference between the vaccine strains and the local pathogenic strains.Therefore,exploring the genetic and antigenic diversity of V.harveyi strains that are prevalent in marine aquaculture regions in our country is of great significance for preparing vaccines for vibriosis occurred in our maricultured species.Based on strains isolated from seven mariculture animals in Zhejiang Province and Fujian Province from 2014 to 2019,this study uses specific PCR for the molecular identification of Vibrio spp.and V.harveyi.In total,123 isolates of Vibrios were identified,of which 54 isolates were V.harveyi.Only V.harveyi isolates were used for further analysis,including genotyping analysis based on intestinal bacteria repeated intergenic consensus sequence PCR(ERIC-PCR),hemolytic analysis,plasmid diversity analysis,antibiotic sensitivity analysis,virulence gene detection.Based on the above analysis results,ten isolates of V.harveyi were selected and analyzed for the virulence in the large yellow croaker(LYC).Three strains were ten chosen to prepare a trivalent inactivated vaccine.The immune effect of the immersion vaccination was analyzed.The main findings of this paper are as follows:(1)The results of the ERIC-PCR molecular typing experiment showed that the 54 isolates of V.harveyi collected from marine farms in the two eastern coastal provinces had a similarity index between 70% and 100%.At 90% similarity level,all isolates could be divided into 12 genotypes.Some isolates obtained from different farms have the same genotype,and are 100% similarity,suggestion the possibility of bacterial spreading between farms.(2)Hemolysis testing results showed that among 54 strains,β-hemolytic(complete hemolysis)strains were 0%,α-hemolytic(incomplete hemolysis)strains were 22.2%,andγ-hemolytic(non-hemolytic)strains were 77.8%.(3)Plasmid profiling results show that 61.1% of V.harveyi(33/54)carried from 1 to 11 plasmids,with the size ranging from 1.3 kb to 30 kb.Interestingly,most(87.5%,14/16)of the isolates recovered from the LYC harbored plasmids(each isolate harbored more than 4plasmids),whereas only 31.6%(7/19)of the isolates obtained from pharaoh cuttlefish carried plasmids(each isolate possessed an average of less than 2 plasmids).(4)The results of the antimicrobial sensitivity testing showed that all analyzed strains were resistant to amoxicillin and ampicillin,but none showed resistance to tetracycline,chloramphenicol,florfenicol,norfloxacin,enrofloxacin and trimethoprim.There was no significant difference in antimicrobial resistance between plasmid-positive strains and negative strains,indicating that the resistance was not encoded in plasmids.(5)The detection of seven typical or atypical virulence genes(chi A,vhp S,vhh,vhp A,vhp B,pap6 and hly A)revealed that all strains were consistent.In total,81.4%(44/54)were positive rate of potential virulence DNA markers(pvm).The virulence analysis results of the 10 selected strains showed that the virulence difference of the strains is not correlated to the above-mentioned genes,but the virulence of the plasmid-positive strains was significantly higher than that of the negative strains(p <0.05).(6)Results of immersion vaccination of the trivalent inactivated vaccine prepared in this study showed that the immune protection was not ideal.According to the V.harveyi-specific antibodies presented in serum and mucus,and expression of immune-related genes,immersion vaccination of the trivalent vaccine could induce the local and systemic adaptive humoral immune responses in large yellow croaker. |
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