Production and purification of heat-stable enterotoxin b from a porcine Escherichia coli strain

Abstract

Production of heat-stable enterotoxin b (STb) by porcine Escherichia coli strains belonging to serogroup 0115 was evaluated in ligated intestinal segments of adult rats. The conditions for optimal production and detection of STb were studied by using the STb-producing strain 4247. As STb production was similar in complex Trypticase soy broth and minimal Davis medium, the latter was used for the fermentation of strain 4247 and the production of STb in large quantities. STb was then purified to apparent homogeneity by sequential ultrafiltration, ultracentrifugation, and preparative gel electrophoresis. The enterotoxin was purified more than 500-fold and exhibited a molecular weight of approximately 5,000 as determined by urea-sodium dodecyl sulfate gel electrophoresis. Purified STb retained such chemical characteristics as resistance to heating (60°C/30 min) and sensitivity to trypsin. A rabbit polyclonal antiserum was produced against the purified toxin. Numerous booster doses were required to obtain a significant enzyme-linked immunosorbent assay titer, suggesting that STb is a poor immunogen. Nevertheless, the antiserum was used successfully to discriminate between culture supernatants of STb-positive and STb-negative 0115 E. coli strains, thus demonstrating the immunogenicity of purified STb.