Document Type

Dissertation - University Access Only

Award Date

1995

Degree Name

Doctor of Philosophy (PhD)

Department / School

Veterinary and Biomedical Sciences

First Advisor

David H. Francis

Abstract

Among ETEC organisms in young pigs, strains expressing K88 fimbrial adhesins are found to be highly pathogenic. Pathogenicity of these E. coli strains depends mainly on adhesion, colonization and enterotoxin production in susceptible pigs. Adhesins confer the adhesive property to these bacteria. Adhesion depends on the presence of specific receptors on brushborder cells of enterocytes. Although the pigs have been phenotyped as A-F based on the adhesion of K88ab, ac and ad variants, localization and distribution of these specific receptors on the intestinal epithelial brush border cells has not been analyzed. In the current investigation, the localization and distribution pattern of receptors for the antigenic variants of K88 (K88ab, K88ac and K88ad) were examined using fluorescence staining of cryostat sections of porcine intestine and isolated brush border enterocytes. Enterocytes were collected from fresh porcine jejunum in nine sequential fractions, from villus tip to crypt base, and cryostat sections from the same tissue were used. Ethanol fixed sections and paraformaldehyde fixed cells were preincubated with biotinylated fimbriae and then FITC conjugated streptavidin. Sections stained with each K88 variant when examined under the fluorescent microscope revealed fluorescent staining on brush borders of the intestinal epithelium from villous tip to crypt base. However, the staining pattern was confluent with K88ab and K88ac in in intestines of pigs of phenotypes A and B, whereas, it was multifocal with K88ad in phenotype A and confluent with phenotype D pigs. Staining of enterocytes from each of the nine fractions indicated no significant variation between the fractions in fimbrial binding. Western blot analysis using solubilized enterocyte fractions and probed with K88ac fimbriae indicated the presence of K88 receptor protein (210 and 240 kD) bands in all nine fractions of phenotype A pigs but not in E. coli infection resistant phenotype E pigs . These results confirmed the presence of receptor through out the villus length to crypt. Since the enterocytes are migratory cells leading to mature cells at villus tips and immature cells at crypt, one can concluded that the K88- receptor expression is not a function of maturity. Challenge of piglets of phenotypes A, C and E with enterotoxigenic E. coli expressing each K88 indicated that, the piglets of phenotype A are more susceptible to K88ab and K88ac, than K88ad. As receptors to K88ab and K88ac appear to be more dense in phenotype A pigs than K88ad, these results suggest that variation in disease susceptibility may be a result of density of the receptors, or the affinity of the binding organisms to these receptors. Monoclonal antibodies were isolated to brush border antigens, and clones were selected that produced antibodies reactive with semipurified K88 receptor glycoproteins. The antibodies from such clones were reactive with brush borders of phenotypes Band F, but not A, C, Dor E. Proteins bound included 210 and 240 kD K88 receptor glycoprotein. It was concluded that K88 receptors in phenotype Band F pigs are structurally different from their in phenotype A animals.

Library of Congress Subject Headings

Escherichia coli infections in swine -- Pathogenesis
Intestines -- Infections
Cell receptors

Format

application/pdf

Publisher

South Dakota State University

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Rights Statement

In Copyright