| Three strains of bacteria were isolated from the diseased Chinese mitten crabs, Eriocheir sinensis, which were cultured in the ponds at Hangzhou. Identification was done, their morphological, physiological and biochemical characteristics of the isolates were the same as those of Vibrio parahemolyticus, therefore, the isolated pathogens from the diseased Eriocheir sinensis belong to Vibrio parahemolyticus. The mortality of artificial infection was 100%, which ascertained that the isolated bacteria were the pathogens of the Eriocheir sinensis. The pathogens were highly sensitive to streptomycin, rifampin, kanamycin, SMZ+TMP, ciprofloxacin, norfioxacin, tetrecycline, ofloxacin, ceftazidime, ceftriaxone, nalidixic acid, et al. The culture suspension of the isolated pathogens was centrifuged, precipitated by (NH4) 2SO4, dialysised, concentrated and filter sterilized, and the extracellular products obtained were found to show gelatinase,chitinase, amylase, caseinase, lipase, phospholipase activities, and to be highly toxic to Eriocheir sinensis.Two kinds of extracellular proteases were purified from the extracellular products of the Vibrio parahemolyticus by ammonium sulfate, Sephadex G-100 and DEAE-Cellulose. The results showed that , the molecular weight of the two kinds of purified extracellular proteases from Vibrio parahemolyticus were 39.6kD and 20kD by SDS-PAGE; the first was stable and displayed high proteolytic activity in the range at temperature of 50 ~ 60℃ and its optimum pH was 9.0; the optimum temperature for the enzyme activity of the latter was 50℃ and the optimum pH was 8.0. EDTA and some metal ions (Cu2+, Mg2+,Fe2+ ) could inhibit the enzyme activity of 39.6kD protease, but Ca2+ was on the contrary that it could activate the enzyme to some extent, so it was suggested a kind of melalloprotease. PMSF could inhibit the enzyme activity of 20kD protease, so it was suggested a kind of serine protease. The two purified proteases were toxic to Vero cells in vitro and cells of the hepatopancreas in Eriocheir sinensis. In this paper, a pair of primers were designed according to the nucleotide sequence of the extracellular proteases of vibrio reported. With the specific primers, one target fragment about 2kb was amplified from the chromosomal DNA of Vibrio parahemolyticus via PCR, which ascertained that Vibrio parahemolyticus isolated from Eriocheir sinensis had extracellular proteases gene in its chromosomal DNA.Isolation was done from the diseased Eriocheir sinensis cultured in the ponds at Hangzhou.Three strains of bacteria were isolated from haemolymph, hepatopancreas, ascites and muscle of the diseased Eriocheir sinensis caused by aeromonas. All of the three isolates were Gram-negative rods, motile by means of single polar flagella, and were facultatively anaerobic; Glucoce was catabolized with production of acid and gas; Oxidase was positive. Two of the three were the same as those of Aeromonas trota in morphological, physiological and biochemical characteristics, therefore, the two pathogens isolated from the diseased Eriocheir sinensis were identified as Aeromonas trota. One of the three was the same as those of Aeromonas hydrophila in morphological, physiological and biochemical characteristics, so, it was identified as Aeromonas hydrophila. The mortality of artificial infection with three isolates were 100%, which ascertained that Aeromonas trota and Aeromonas hydrohila were the pathogens of the Eriocheir sinensis. Both of Aeromonas trota and Aeromonas hydrophila isolated from diseased Eriocheir sinensis could produced pathogenic factors such as outer membrane proteins, extracellular proteases and hemolysin.Synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) technique to amplify a sequence of the aerA gene, which encoded the aerolysin of Aeromonas trota. A DNA fragment of 622bp was amplified from Aeromonas trota isolated from Eriocheir sinensis. The fragment was digested with BamHI restriction endonuclease and produced 570bp and 50bp fragment. Thi... |
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