Purification and characterization of toxins from Aeromonas hydrophila virulence strain from hybrid catfish (Clarias gariepinus X Clarias macrocephalus)
1998
Thapanee Sirirat
Purification of cytolytic enterotoxin from A. hydrophila which their LD50 values (96 hrs.) were 2.33*10*[5), 2.84*10*[5), 5.41*10*[7), 9.61*10*[7), 1.18*10*[8), 1.40*10*[8) and 3.54*10*[8) CFU/ml by filtration with ammonium sulfate, purification with hydrophobic column chromatography on Econo-Pac HIC Cartridge and anion exchange chromatography on DEAE-Bio-Gel A. The protein composition of cytolytic enterotoxin was studied by using SDS-PAGE. The result showed that the extracts had 52 kDa protein as a major component. The highly virulent strains with an LD50 of approximately 10*[5) cell/ml had higher quantity of cytolytic enterotoxin-silver stained band than the low virulent strains with and LD50 between 10*[7)-10*[8) CFU/ml. To study antibody production, hybrid catfish and rabbit were immunized with cytolytic enterotoxin extracts from virulent strain of A. hydrophila. Antibodies against cytolytic enterotoxin were detected by indirect ELISA. The results from indirect ELISA showed that hybrid catfish and rabbit were developed a high antibody against cytolytic enterotoxin which their antibody titres were 1:1,024 and 1:32,768 respectively. The specific antigenic protein of cytolytic enterotoxin were recognised using Western blot analysis. The result demonstrated that the antibody from hybrid catfish was specific with 52 kDa. It indicated that cytolytic enterotoxin is an antigenic protein of A. hydrophila.
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