碩士 / 國立臺灣海洋大學 / 水產養殖學系 / 104 / This study investigated the pathogenicity and virulence factors of Vibrio strains Lvhpvpa、VnLvvpa 2、Abvpa1 and HlLvvpa isolated from white shrimp (Litopenaeus vannamei) and abalone (Haliotis diversicolor supertexta). After detection of biochemical characteristics and specific genes etc, these Vibrio strains were confirmed to be Vibrio parahaemolyticus. In addition, after checking by the presence of specific genes of AHPND strain, Lvhpvpa and Abvpa1 were identified as AHPND strains, and VnLvvpa 2 and HlLvvpa as Non-AHPND strains. All the four strains had same results of biochemical characteristics, and were susceptible to Flumequine, Florfenicol, Doxycycline and Oxolinic acid. The protease activities in the ECP of strains Lvhpvpa、VnLvvpa 2 and HlLvvpa could be inhibited by the addition of 1,10-phenanthroline monohydrate、PMSF and Pepstatin A. Only strain Abvpa 1 could not be completely inhibited by PMSF and Pepstatin A. The above results indicated that there were metallopase、serine and acid proteases in both types of V. parahaemolyticus. The PLC activities in the ECP of the four strains could be inhibited by phospholipase C inhibitor Compound 48/80. The relative activity of protease of four strains could reach to 100％~110％ under pH 7~pH 10, and the relative activity of PLC of four strains were increased as pH rised. Erythrocytes of tilapias and groupers could be utilized by the four strains, with higher hemolytic activities detected in AHPND strain compared to that of Non-AHPND strains.
In toxicity test, the LD50 of bacteria of AHPND strain Lvhpvpa and Non-AHPND strain VnLvvpa 2 for white shrimp were 2.32×104 and 2.15×105 CFU/g shrimp body weight, respectively. The LD50 of ECP of AHPND strain Lvhpvpa and Non-AHPND strain VnLvvpa 2 for white shrimp were 0.128 and 1.031 μg protein/g shrimp body weight, respectively. The addition of 1,10-phenanthroline monohydrate、PMSF and Pepstatin A obviously inhibited the lethal toxicity of the ECP of AHPND strain Lvhpvpa and Non-AHPND strain VnLvvpa 2. The addition of Compound 48/80 could not completely inhibit the toxicity of ECP from both strains. From a series of inhibitor related tests, it seemed that proteases could be the main toxins of V. parahaemolyticus, and of those the metalloprotease was suggested to be more important.
For alpha-actin gene test in white shrimp, the gene expression of Non-AHPND strain VnLvvpa 2 was significantly higher than that of AHPND strain Lvhpvpa, while there were no significant difference between control and test groups for beta-actin gene expression, after injecting with respective ECP. After intramuscular injection challenge with AHPND strain Lvhpvpa and Abvpa 1 ECP, they could be observed AHPND symptom through histopathology.
The present results revealed that the virulence and hemolytic activity of AHPND strains were stronger than that of Non-AHPND strains. In addition, a pathogenic strain isolated from Taiwan small abalone was for the first time identified as an AHPND strain. The proteases of both types of V. parahaemolyticus were suggested as important virulence factors, especially metalloprotease and phospholipase C, and they may act synergistically. The presence of PirAB-like proteins in AHPND strains may enhance lethality in white shrimps.
| Identifer | oai:union.ndltd.org:TW/104NTOU5086032 |
| Date | January 2016 |
| Creators | Liu, Pei-Wen, 劉姵妏 |
| Contributors | Liu, Ping-Chung, Lee, Kuo-Kau, 劉秉忠, 李國誥 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 81 |
Page generated in 0.0071 seconds