Prevalence Characteristic And Pathogenicity Difference Analysis Of Vibrio Harveyi Isolated From Aquaculture Animals

Vibrio harveyi is known to be on a major pathogenic vibrio in mariculture animal.Disease outbreaks attributed to V. harveyi has been reported in variety of aquatic animal farm production. In recent years,mass mortalities of many cultured kinds of maricultured fishes,such as P. vannamei and Lutjanus sanguineus have occurred frequently infected by V. harveyi. However for knowing the disease cause by V. harveyi time is short and its epidemiological studies is not deeply enough, especially in domestic lack of V. harveyi molecular type analysis research. In this study, we isolated 57 strains V.harveyi from the diseased mariculture animals from the Fujian, Jiangsu,Zhejiang and Hainan province by PCR molecular identification biochemistry identification system.A molecular classification by using the methods of pulsed-field gelelectrophoresis(PFGE)was performed to investigate the variability among 57 V. harveyi strains,and then a comparative study on pathogenicity and biochemical characteristics of ten V.harveyi isolates were carried out,including bacterial infection experiment,antimicrobial susceptibility,biochemical characteristics,main virulence factors detection and secretory proteins difference analysis of islates in different virulence.The most results on were as follow:1、PFGE results:54 strains typed were divided into 38 kinds of different patterns with similarity range:36%-100% through the method of Pulsed Field Gel Electrophoresis.Different areas had different kinds of PFGE patterns:except strains from Jiangsu and Hainan were divided into 11 kinds of patterns respectively,Zhejiang had 7 kinds of patterns,while Fujian had 8.There were 1-4 dominant patterns in one area.Overall, There was generally closer genetic relationship between Vibrio harveyi isolates from the same region,while strains were generally quite different from different regions;strains of each region belonged to multi-patterns,and the patterns were dispersive,which indicated that presently,multiple Vibrio harveyi clones were only popular in regions respectively,and prevolence range between different regions was not wide.2、The results of pathogenicity and biochemical characteristics of ten Vi brio harveyi isolates:Ten V.harveyi strains(Vh00947,Vh00949,Vh11011,Vh11014, Vh21217, Vh21218, Vh21220,Vh21229,Vh21231 and Vh31487)were isolated and identified from Penaeusvannamei cultured in the South of China. By bacterial infection experiment and detection of main virulence factors of V. harveyi, their virulences and possible causative agents associated with which were explored.The results showed that, except for the isolates Vh21229 and Vh00949, all of the strains were highly virulent.Biochemical characteristics test showed that the strain Vh21229 was different with the other strains on the four test items D-mannose, sucrose, D-trehalose and lysine decarboxylase, suggesting that vibrios with different virulence might have different biochemical characteristics. Furthemore, eight main virulence genes of V.harveyi including the hemolysin gene Vh1, Vh2, Vh3 and Vh4, the vibrio virulence regulation gene tox S, and virulence protein gene Hcp, Zot and Pap6 were detected by PCR,and the result indicated that gene Pap6,tox S and 4 hemolysin genes were rarely detected in all of the strains, while the detection rates of gene Hcp and Zot in virulent strains were respectively equal or higher than their total detection rates, demonstrating that the two virulence genes Hcp and Zot were closely correlated with the degree of pathogenicity of V. harveyi.The antimicrobial susceptibility test showed that there were no significant differences among the 10 strains on MIC values of 16 common drugs. However, it is notably that all of the strains were strongly resistant to the antimicrobial agenterythromycin and ampicillin,suggesting the use of antibiotics should be managed and controlled strictly in shrimp farms.And secreted extracellular proteins analysis was carried out by SDS-PAGE and mass spectrometry in virulent strains Vh21220, Vh31487 and attenuated strain Vh21229.The result showed that outer membrane protein(Omp U) were highly expressed in Vh21220(NW 36.4KDa,PI 4.26) and Vh31487(NW 35.8KDa,PI 4.19),while aminopeptidase in Vh21229(NW 65.5Kda,PI 5.32).It indicated that pathogenic role of outer membrane protein on the host was stronger than aminopeptidase.