ELISA TO DETERMINE ANTI-K99 PILUS ANTIBODY IN THE SERA OF NORMAL AND DIARRHEIC CALVES

An ELISA to detect circulating antibodies against K99 pili, a major at tachment factor to intestinal epithelial cells of Escherichia coli in calves, was performed. Two methods of K99 pili purification were attem pted. Best results in terms of purity of the K99 antigen were achieved following the method described by Karkhanis and Bhogal (1986). This p rocedure included a heat shock at 65 degrees C during 25 min to releas e the pili and ultracentrifugation steps to purify the antigen. SDS-PA GE showed an 18 KDa major band, identified as the K99 pilus antigen af ter immunoblotting against reference antisera. The purified K99 antige n was then adsorbed to the ELISA microplates. High optical density was obtained in the ELISA using a pool of sera from immunized cows. No di fferences in antibody levels (P greater than or equal to 0.05) could b e detected between clinically healthy calves and those showing diarrho ea.