The Toxicity Of Vibrio Vulnificus Isolated From Aquatic Products And The Change Of Its Cell Ultrastructure And Toxicity Under The Treatment Of Ultrahigh Pressure

In the present study, Vibrio vulnificus (V.vulnificus) isolated from aquatic product have been evaluated about toxicity by hemolysis test and acute toxicity, compared with standard V.vulnificus strain CICC21615. It has been confirmed through biochemistry tests, cytolysin gene amplification and gene sequencing that six suspected strains isolated from aquatic product are all V.vulnificus strains, except from No.2isolated strain. Six isolated strains have all amplified gene bands about222bp; while CICC21615V.vulnificus strain has amplified gene bands about400bp.In the hemolysis test, the ratio of diameter between hemolysis circle and colony of standard strain is3, while the ratio of isolated strains are2.3,2.0,2.8,2.0,2.5and2.1from No.1strain to No.6strain, respectively. In the test of acute toxicity, the LD50of standard strain is3.776×107CFU/mL, while the LD50of No.3strain is2.218×108CFU/mL. In conclusion, standard V.vulnificus strain CICC21615is moderate virulent strain, while No.3isolated V.vulnificus strain is low virulent strain.Differences of toxicity and viscera toxic effects in mice between V.vulnificus cd03and V.vulnificus CICC21615have been studied through acute toxicity test. The mice were injected with V.vulnificus CICC21615and V.vulnificus cd03of concentration of bacteria liquid of106CFU/mL,107CFU/mL and108CFU/mL in enterocoelia respectively, and the injection dose was0.02mL/g. By intraperitoneal injection of mice after24h, The dose group of108CFU/mL of liver coefficient of V.vulnificus CICC21615and V.vulnificus cd03reached7.30%and7.30%, respectively, and were higher than other dose group of107CFU/mL,106CFU/mL and the control group, significantly. The vacuoles of liver cell of mice injected with two strains of vibrio vulnificus of108CFU/mL dose groups had been degenerated, with the damage of internal structure and the rupture of cell membrane. The intercellular space in the splenic tissue increased in the dose group of108CFU/mL, and it appeared a lot of white spots, the spleen organization was obviously loose. The dose groups of108CFU/mL of V.vulnificus CICC21615and V.vulnificus cd03, the addition of GOT, GPT and the reduction of GST were up to16.62and11.62,5.77and5.6,46.25and37.29U/gprot, significantly. The content of MDA was not changed significantly among all dose groups. In the infected mice, vibrio vulnificus were mainly distributed in the liver and spleen, had a small amount of distribution in the kidney, and were not isolated any single colony in the blood. The results of these tests show that viscera toxicity of V.vulnificus CICC21615is more acute than the one of V.vulnificus cd03. Damage of the liver and spleen is more and more aggravating with the increase of concentrations of bacteria liquid, and high concentration of bacteria liquid can not only accelerate the damage of liver tissue, but also improve the speed of the self-repairment of cells. It may activate and accelerate the internal related damage renovation in the cells.Through the ultrahigh pressure processing experiment, the change of ultrastructure and toxicity of the cells of Vibrio vulnificus isolated from aquatic product had been studied. V.vulnificus CICC21615and V.vulnificus Cd03under the treatment of ultrahigh pressure of100,150,200,150,200mpa had been studied about bacteria survive counts, observation from transmission electron microscope, determination of ultraviolet absorption of260nm and280nm, hemolysis test and acute toxicity test of mouse, untreated group as control group. V.vulnificus CICC21615under the200mpa pressure, only had5single colony, and the fatality rate had close to100%. When V.vulnificus CICC21615treated under more than250mpa or250mpa, the fatality rate was100%, V.vulnificus Cd03fatality rate was100%when V.vulnificus Cd03treated under more than200mpa or200mpa. cell wall structure of vibrio vulnificus of Control group was intact, distribution of cytoplasm was in order. With the increase of pressure, the cell walls had shrink significantly. At the same time the membrane had damaged obviously and had rupture partially, with the leakage of inclusions and the occurrence of penetration of large-area electronic area. When Vibrio vulnificus treated under more than200mpa or200mpa, cell membrane of Vibrio vulnificus had disappeared, with the polymerization of cell nuclear and the condensation of bacterial cell protein. In the ultraviolet absorption of260nm, V.vulnificus CICC21615and V.vulnificus Cd03reached the top at250mpa and200mpa, respectively, and had reached0.105and0.119respectively; In the ultraviolet absorption of280nm, V.vulnificus CICC21615and V.vulnificus Cd3mpa both reached the top at250, and had reached0.106and0.187respectively. With the increase of pressure, hemolysis activity and acute toxicity of V.vulnificus CICC21615and V.vulnificus Cd03decreased constantly, and when treated under more than200mpa or200mpa the hemolysis activity reduced to0. when treated under more than150mpa, V.vulnificus CICC21615and V.vulnificus Cd03both could not make mouse to death. When V.vulnificus Cd03treated with lower pressure (200mpa), the fatality rate can reach100%. the reasons of death of bacteria under ultrahigh pressure had associated with the damage of cell membrane, and also associated with the leakage of nucleic acids and proteins. The aggregation of proposed nuclear and active protein is also the important reason for the death. Experimental research also found that with the increase of pressure, hemolysis activity and acute toxicity of strain were significantly decreased. For it, the direct reason had been related to the number of living bacterium under different pressure processing.