Studies on the Virulence and Biochemical Characterization of the Extracellular Products of Aeromonas hydrophila in Soft-shelled Turtle (Trionyx sinensis)

• Main Authors: SUN KUANG-PING, 孫廣平
• Other Authors: Tsai S. S.
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/29470912011933886965

碩士 === 國立屏東科技大學 === 獸醫學系 === 92 === Although Aeromonas hydrophila has been considered as an opportunist or secondary pathogen in stressed animals or immunocompromised hosts, it is recognized as an important pathogenic microorganism for hemorrhagic septicemia of soft-shelled turtle. The clinical signs of infected soft-shelled turtles are lack of appetite, swollen and weakness of neck, hemorrhages in oral and nasal cavities, breastplate, muscular edema, hemorrhagic gastro-enteritis and septicemia. Infection by this microorganism can lead into a serious mortality and thus causes a great economic loss in the soft-shelled turtle industry. Ten strains of A. hydrophila were randomly selected from the Southern Regional Fish Disease Center during 1999 to 2002, together with the type strain of A. hydrophila (ATCC 7966) for confirmation study. The conventional morphological and biochemical methods as well as API 20NE system and Biolog GN2 system were used to identify of these strains. In this study the virulence factor of A. hydrophila to soft-shelled turtle and its biochemical characterization were studied. The strain AR-01-359 of A. hydrophila was chosen as a candidate due to it produced the most serious symptoms and lesions by pathogenicity screening test. The 50 percent lethal dose (LD50) of the live bacterial suspension, the crude extracellular product (ECP), and the ammonium sulfate purified ECP were 1.04×104 CFU/ g (body weight, BW), 5.952μg/ g (BW) and 3.08μg/ g (BW) of soft-shelled turtles, respectively. The crude ammonium sulfate precipitated ECP was further purified by various steps of hydrophobic interaction chromatography and anion exchange chromatography. The molecular mass of the final purified product was 28 kDa as estimated by SDS-PAGE. The resultant product was identified as a protease with acetylcholinesterase and caseinase activities. It was inhibited by EDTA-2Na (ethylenediamine tetraacetic acid disodium salts), CaCl2, HgCl2, ZnCl2 and KMnO4, thus it was further concluded as a metalloprotease. This purified protease showed the maximal activity at pH 10 and 70 ℃, and was lethal to soft-shelled turtle with 3.67 μg protein/g (BW). The hemolytic activities of this protease against erythrocytes of soft-shelled turtle, tilapia, largemouth bass, sheep and rabbit were 109, 218, 435, 218 and 871 units/ mg protein, respectively. Results of the rabbit ileal loop assay were demonstrated that there were appositive relationship between the severity and the dosages in the hemorrhagic intestinal lesions.