Document Type

Thesis - University Access Only

Award Date

2009

Degree Name

Master of Science (MS)

Department / School

Veterinary and Biomedical Sciences

First Advisor

Weiping Zhang

Abstract

Enterotoxigenic Escherichia coli (ETEC) strains expressing K88 fimbriae and LT or/and ST enterotoxins are a major cause of diarrhea in neonatal and weaned pigs. ETEC diarrhea causes substantial economic loss to swine producers. Thus, developing prevention strategies against this disease is a great need. However, conventional vaccines including a single antigen from either fimbriae or enterotoxins are not effective. An effective vaccine should include antigens from multiple virulence determinants. The objective of this study is to develop a vaccine platform by using K88ac (F4ac) fimbria to express enterotoxin antigens. By replacing two epitopes (EP3 and EP4) of the FaeG major subunit of the K88ac fimbria with the LTB and/or STa epitope(s), we constructed four strains expressing chimeric fimbriae: 8551 (K88ac:LTB3), 8549 (K88ac:LTB4), 8576 (K88ac:STa3:LTB4), and 8616 (K88ac:STa3). Expression of the chimeric K88ac fimbriae, LTB and STa antigens in these constructs was confirmed. Four E. coli strains expressing 6xHis tagged chimeric FaeG protein were constructed, and purified chimeric FaeG proteins were used to vaccinate adult rabbits. Strong humoral immune responses (anti-K88ac, anti-STa, and anti-LT antibodies) were detected. Bacteria adherence blocking assay indicated that adherence of wild type ETEC strain 3030-2 to IPEC-J2 cells was block by antibodies from rabbit sera and some fecal samples. Antibody neutralization assay using cyclic AMP EIA and cyclic GMP EIA suggested that anti-LT and anti-STa antibodies were protective in vitro as they could reduce the toxicity of cholera toxin and STa toxin to T84 cells. This study demonstrates that the K88ac vi fimbria can be served as a platform to express enterotoxin antigens to induce protective antibodies against both fimbria and enterotoxin antigens. It suggests that K88ac fimbriae could be an excellent platform to express additional antigens for developing multivalent vaccines against porcine ETEC diarrheal disease. It may also provide instructive information for developing vaccines against other enteric pathogens. In the second part of this thesis, we investigated genetic heterogeneity of the LT and STa genes of ETEC strains isolated from diarrheal pigs. We sequenced the eltAB gene encoding LT toxin from 52 porcine ETEC strains and estA gene encoding STa toxin from 33 porcine ETEC strains. DNA sequence data showed the LT and STa genes isolated from porcine ETEC were porcine LT and porcine STa exclusively. Furthermore, all pSTa and over 90% of the pLT gene sequences were identical. Only four (out of 52) ETEC strains showed heterogeneity of LT gene sequences. To evaluate the impact of these two mutations on LT binding activity to the host receptor GMI, we constructed three isogenic strains: 8458 (pLT, wild-type), 8647 (S44N in LTB) and 8649 (S60T in LTB), and mutations at the 44 th and the 60th amino acid of pL TB subunit were shown to significantly reduce the GMI-binding affinity and toxicity.

Library of Congress Subject Headings

Escherichia coli infections in swine
Escherichia coli
Swine -- Diseases
Vaccines
Enterotoxins

Publisher

South Dakota State University

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Rights Statement

In Copyright