A PCR-based method of detection and differentiation of K88+ adhesive Escherichia coli.
The objective of this study was to develop a polymerase chain reaction (PCR)-based method to detect and differentiate among Escherichia coli strains containing genes for the expression of 3 antigenic variants of the fimbrial adhesin K88 (K88ab, K88ac, and K88ad). Five primers were designed that allowed detection of K88+ E. coli, regardless of antigenic variant, and the separate detection of the ab, ac, and ad variants. Primers AM005 and AM006 are 21 base pair (bp) oligomers that correspond to a region of the K88 operon that is common to all 3 antigenic variants. Primers MF007, MF008, and MF009 are 24-bp oligomers that matched variable regions specific to ab, ac, and ad, respectively. Using primers AM005 and AM006, a PCR product was obtained that corresponds to a 764-bp region within the large structural subunit of the K88 operon common to all 3 antigenic variants. Primer AM005 used with MF007, MF008, or MF009 produced PCR products approximately 500-bp in length from within the large structural subunit of the K88 operon of the 3 respective antigenic variants. Fragments were identified by rates of migration on a 1% agarose gel relative to each other as well as to BstEII-digested lambda fragments. This PCR-based method was comparable to the enzyme-linked immunosorbent assay and western blot test in the ability to differentiate between the antigenic variants. K88+ E. coli were differentiated from among laboratory strains and detected in ileal samples taken from cannulated pigs challenged with a known K88+ variant. K88+ E. coli were also detected from fecal swabs taken from newly weaned pigs, thus confirming that this PCR-based test could provide a convenient clinical assay for the detection of K88+ E. coli. Detection and differentiation of K88+ E. coli using general and specific primers was successful. PCR methods of detection should permit identification of K88+ antigenic variants regardless of the level of expression of the antigen.
Franklin
M A
MA
Department of Animal Science, University of Tennessee, Knoxville 37901, USA.
Francis
D H
DH
Baker
D
D
Mathew
A G
AG
D018830:Adhesins, Escherichia coli / Q000235:genetics*; D000818:Animals; D001422:Bacterial Adhesion; D015153:Blotting, Western / Q000379:methods; D017931:DNA Primers; D003967:Diarrhea / Q000382:microbiology / Q000662:veterinary; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D004927:Escherichia coli Infections / Q000175:diagnosis / Q000662:veterinary; D014644:Genetic Variation; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D013552:Swine; D013553:Swine Diseases
D018830:Adhesins, Escherichia coli; D017931:DNA Primers