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Pathogenicity Research of the Pathogen Associated with Ulceration in Cultured Sea Cucumber (Apostichopus Japonicus)

Author: YangJiaLong
Tutor: ZhouLi;ZhanWenBin
School: Ocean University of China
Course: Aquaculture
Keywords: The sea cucumber ulcer disease Kill salmon Aeromonas Pathogenicity Extracellular products Extracellular protease Virulence factors
CLC: S947.9
Type: Master's thesis
Year: 2008
Downloads: 169
Quote: 1
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Abstract


Japonicus (Apostichopus japonicus) is nourishing and medicinal boutique, in China's Shandong and Liaoning provinces in the late 1990s set off a craze of farmed sea cucumber, sea cucumber has become the emerging farming promote the development of China's northern coastal aquaculture varieties. With the increasing scale of farming, sea cucumber diseases are also increasingly significant, since 2003, around the breeding sea cucumber frequency of ulcer disease, has seriously hampered the rapid development of sea cucumber aquaculture. Therefore sea cucumber ulcer disease epidemiology and pathogenesis research become an urgent issue. In this study, the risk of ulcer disease sea cucumber lesions isolated two dominant bacteria (H1 RH2), artificial infection experiments confirmed that the two strains were pathogenic, the H1 virulent strains, Rh2 for the force attenuated strains, and On this basis, the two pathogens were identified. Extracellular products (extracellular products, ECP), and the pathogenicity of the ECP extract virulent strain H1, containing the types of enzymes, enzyme activity, protein composition and antigenic characteristics were analyzed. Gel exclusion chromatography and anion exchange chromatography separation and purification of a serine protease, pathogenicity experiments from the ECP that it is the strain H1 virulence factors, from biological enzymatic and proteomics point of view of the protease characteristics were analyzed. The specific methods and results are as follows: March 2005, Shandong Rushan sea cucumber farms large-scale stichopus ulcer disease, symptoms of anorexia, surface ulcers, swollen mouth, spit the intestines, and finally death autolysis. Suffering from canker the sea cucumber lesions separated into two dominant bacteria (H1 and RH2) with bath, four kinds of trauma bath, body cavity injection and intramuscular injection of the body wall of sea cucumber infection, the results prove that the two strains are pathogenic, H1 virulent strains, by the the body the wall trauma bath and body wall intramuscular infection sea cucumber, and the water temperature is low virulent, lethal concentration of sea cucumber ( LD50) of up to of 1.4 × 106CFU / Last; Rh2 Attenuated Strains, only to body wall intramuscular infection sea cucumber, sea cucumber median lethal concentration (LD50) of of 5.7 × 106CFU / tail. Conventional physiological and biochemical indicators identified strains H1 kill Aeromonas salmonicida bacteria to kill Japanese salmon subspecies (Aeromonas salmonicida masoucida) strain RH2 as Vibrio alginolyticus (Vibrio alginolyticus); determined on the basis of the 16S rRNA gene sequence of the strain RH2 and heat shock protein HSP60 (Heat shock protein HSP) gene sequences in the NCBI homology search and phylogenetic analysis, results showed that the development the strains RH2 V.alginolyticu tree system built in the 16S rRNA gene ( the sAF513447) natural aggregate a confidence level of 50%, and in HSP60 gene construct system development strains RH2 V.alginolyticus (AY332570, AF230931) clustered into a branch of the tree, the confidence level of 99%. Cellophane plate method to extract the virulent strain H1 extracellular products (ECP), which can cause the sea cucumber death, similar to the onset of symptoms and nature, the sea cucumber semi-lethal dose of 5.24μg protein / g body weight; ECP has Casein, gelatin protease, chitinase and amylase activity and hemolytic; the case on the substrate azocasin role to detect the protease activity of the ECP, the role of a temperature of 25 ° C when the enzyme activity of 470.6 activity unit / mg protein, with optimum substrate temperature to 50 ° C, and thermally unstable, when the temperature exceeds 40 ° C, the enzyme activity showed a downward trend, more than 70 ℃ The enzyme is inactivated. ECP structure were analyzed by SDS-PAGE, the protein composition ECP about 13 protein bands, of which the principal with 9; using mouse anti-ECP serum Western-blot analysis, the results showed 13 protein bands of the ECP 7 immunogenic, can stimulate the body to produce immune responses secrete antibodies. Virulence factors in the ECP was purified using Sephadex G-100 gel exclusion chromatography and DEAE anion exchange chromatography, to obtain a molecular weight of 42 kDa protease capable caused stichopus death, symptoms associated with strain H1 initiator symptoms of stichopus death similar, sea cucumber semi-lethal dose of 1.12μg / g body weight, which is 4.7 times the ECP virulence, indicating that the protease strain H1 virulence factors. From culture supernatant, after a few steps purified protease yield of 1.7%, and the enzyme specific activity increased by 21.9 times. The protease substrate optimum of 40 ℃, the optimum pH was 8.0, the thermal instability, when 70 ° C and he was inactivated; protease 5 mM serine protease inhibitor PMSF 98% inhibition of enzyme activity , Ca2 and Mg2 can be increased by 9% and 3% of the enzyme activity. Determination of protease N-terminal 15 amino acid residue sequence, NCBI homology search result display with the highest homology to serine protease. Protease two-dimensional electrophoresis molecular weight of 42 kDa protease that alkaline two proteins by isoelectric points of composition.

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CLC: > Agricultural Sciences > Aquaculture, fisheries > Fisheries Protection > Other aquatic pests and diseases and their prevention > Other
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