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. / Graduation date: 1992
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/36589 |
| Date | 11 February 1992 |
| Creators | Rutherford, Gregory J. |
| Contributors | Matsumoto, Maskazu |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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