Document Type
Thesis - University Access Only
Award Date
2009
Degree Name
Doctor of Philosophy (PhD)
Department / School
Biology
Abstract
Enterotoxigenic Escherichia coli (ETEC) are the most common bacterial cause of dehydrating diarrhea in humans and animals worldwide. Porcine enteric colibacillosis has caused major economic losses in the farming industry due to increased mortality, reduced production efficiency and increased treatment costs. It has been well recognized that colonization of the small intestine of neonatal and early weaned pigs by ETEC accounts for most gastrointestinal disorders in these animals. Past studies have demonstrated that the most common and severe porcine ETEC infections are caused by strains that express K88 (F4) fimbriae, heat-labile enterotoxin (LT), and heat-stable enterotoxin b (STb). Recent studies demonstrated that expression of LT enhanced colonization of gnotobiotic piglets by adhesin-producing E. coli. Wild-type ETEC adhered in significantly greater numbers to enterocytes than did non-toxigenic constructs. In the present study, the contribution of LT to ETEC adherence on intestinal cells was quantified by conducting adherence inhibition assays utilizing the IPEC-J2 cell line and modified E. coli strain 1836-2 (K88ac + adhesin) expressing either the LT holotoxin (strain 8035), a catalytically inactive form of LT [LT(Rl 92G); strain 8221 ], or LTb subunits only (strain 8589). Isogenic LT strains 8035, 8221, and 8589 were able to adhere in significantly higher numbers to IPEC-J2 cells than strain 8017 (K88ac + pBR322 control). In addition, isogenic strains 8035, 8221, and 8589 substantially inhibited virulent ETEC strain 3030-2 (K88+/astA +/LT+/STb +) adherence in blocking assays, while strain 8017 which did not express LT was unable to reduce adherence of 3030-2 to the IPEC-12 cells. To further test the ability of non-pathogenic ETEC constructs to colonize the intestine and competitively exclude ETEC at the level required to produce clinical disease, we utilized a piglet ETEC challenge model. Five-day-old piglets were inoculated with either a placebo (negative control group) or with any of the three K88+ E. coli strains with modified LT expression: strains 8017, 8221, or 8488 (LT(R192G) gene fused to the gene for STb in pBR322). Piglets were challenged with virulent ETEC strain 3030- 2, 24h after piglets were inoculated with one of the mutated strains. All K88ac receptorpositive piglets in the negative control group developed diarrhea and became dehydrated 12 hours post-challenge. Piglets inoculated with strain 8221 or 8488 did not exhibit any clinical signs of ETEC disease while piglets inoculated with ETEC strain 801 7 showed mild diarrhea to no diarrhea post-challenge. There was significant weight loss in the control pigs compared to the piglets inoculated with the isogenic LT strains and blood total protein was significantly higher in the control pigs compared to the pigs inoculated with strains 8221 or 8488. Quantitative culture of the challenge strain in washed ileum and jejunum indicated a significant higher number of pathogenic E. coli in pigs of the control group compared to pigs in groups inoculated with strains 8017 or 8488 for both segments of the small intestine. There was a significantly higher number of pathogenic E. coli in the ileums of pigs inoculated with strain 8017 compared to the 8488 group. The most common adhesin of porcine ETEC is K88 (F4). The 3 serological antigenic variants of K88 are: K88ab, K88ac, and K88ad; however, K88ac is the common variant expressed on ETEC strains isolated from diarrheic pigs. Susceptibility of piglets to K88ac + ETEC has been correlated with the adherence of bacteria to isolated enterocyte brush borders and expression of a porcine intestinal brush border mucin-type glycoprotein (IMTGP) which binds K88ab + and K88ac + ETEC. Recently, a DNA-marker based test, the PCR-restriction fragment length polymorphism (PCR-RFLP) test, was developed to allow for genotyping for ETEC K88ab/ac resistance/susceptibility. We wanted to observe the relationship between the PCR-RFLP test, the intestinal brush border assay, and the brush border analysis for the presence of IMTGP to piglet susceptibility/resistance to K88ac. Based on our observations, IMTGP had the strongest correlation among the test compared to piglet susceptibility/resistance to K88ac. Although PCR-RFLP does not have high accuracy and reliability, it is a convenient first screening assay which allows screening of piglet susceptibility towards K88ac prior to K88ab/ac ETEC experimentation.
Library of Congress Subject Headings
Escherichia coli infections in swine
Enterotoxins
Escherichia coli
Format
application/pdf
Number of Pages
146
Publisher
South Dakota State University
Recommended Citation
Santiago-Mateo, Kristina B., "The Role of Heat-Labile Enterotoxin in the Colonization of the Small Intestine by Enterotoxigenic Escherichia Coli" (2009). Electronic Theses and Dissertations. 1612.
https://openprairie.sdstate.edu/etd2/1612
