Development of an enzyme-linked immunosorbent assay to detect antibodies against Bacillus thuringiensis subspecies israelensis in Mallard ducks (Anas platyrhynchos) Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/b5644w814

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  • To develop an assay to detect antibodies to Bacillus thuringiensis subsp. israelensis in mallard ducks, a growth curve was first established for the bacterium. The growth curve indicated that the crystal delta endotoxin would be best harvested from the rest of the cell material after 12 hours of growth. The delta endotoxin was solubilized in alkaline conditions followed by treatment with proteases or no treatment. The two differently treated delta endotoxins were purified by column chromatography. Fractions were assayed for duck erythrocyte lysis and cytotoxicity to a mosquito cell line. The proteolyzed sample gave four protein peaks with gel filtration, and the fourth peak containing biological activity was further separated into three protein fractions by anion exchange chromatography; two of the three showed biological activity. These two fractions contained 22 and 23 kD proteins species. The nonproteolyzed sample was separated into two protein fractions by gel filtration; only the first peak contained the biological activity. This fraction was further separated into two fractions by anion exchange chromatography; only the second fraction, containing a 28 kD protein, exhibited the activity. This fraction contained a 28 kD protein. However, the fractions containing 22 or 23 kD proteins originating from the proteolyzed sample showed the highest biological activity. Mallard ducks were repeatedly exposed to an aerosolized commercial preparation of the organism. Sera were collected periodically and tested for the antibody by an enzyme-linked immunosorbent assay (ELISA). Those toxic antigens containing 22 or 23 kD proteins were unsuitable for the assay. The exposed ducks were found to produce antibodies against the first fraction from anion exchange chromatography of the proteolyzed sample. The antibody titres increased as the number of exposures increased. The results suggest that ELISA is applicable for detecting antibodies against B.t.i. in wild ducks using the fraction containing a 50 kD protein.
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