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The complexity of the small intestinal F4 Escherichia coli receptors in pigs and its role in immunization

Ut Nguyen Van (2015)
abstract
Post-weaning diarrhea is mainly caused by F4+ enterotoxigenic Escherichia coli (F4+ ETEC), leading to severe economic losses. Three different F4 variants exist, F4ab, F4ac and F4ad, with pigs showing adhesion of one or more of these variants or completely F4 receptor negative (F4R-). For long-term management of the infection, selection for F4R- pigs and/or vaccination of F4R+ pigs seems to be the best strategies to prevent postweaning diarrhoea. However, neither the causal mutation resulting in F4+ ETEC resistance nor the biochemical nature of the F4R have been determined yet. This thesis aimed to characterize and identify F4R glycoprotein(s). In the first study, we evaluated the prevalence of the F4+ ETEC susceptible phenotypes among Flemish pig breeds using an in vitro villous adhesion assay. Based on adhesion of the 3 F4 variants, seven different F4+ ETEC susceptible phenotypes were found. Overall, F4ab and F4ac E. coli adhered more numerous to intestinal villi than F4ad E. coli. The completely resistant phenotype E was found in 24.08% of the pigs. F4acR+ pigs can be orally immunized with F4ac fimbriae, one of the criteria used to select pigs for F4R glycoprotein characterisation. However, most pigs have F4ac-specific maternal antibodies, which mask the serum antibody response upon oral immunization. Therefore, we established in the second study an ELIspot test to detect the antibody response in piglets having colostral F4ac-specific serum antibodies. This test allowed us to identify circulating F4ac-specific antibody secreting cells (ASCs). Total F4ac-specific ASCs as well as IgA F4ac-specific ASCs revealed an F4ac-specific antibody response in the orally immunized animals with maternal antibodies in the absence of an increase in serum antibodies. Interestingly, the IgA F4ac-specific ASC response on enriched IgA+ B cells elucidated a more robust IgA booster response upon oral immunization of pigs with than without maternal antibodies. To select potential intestinal F4R glycoproteins, we determined in the third study the binding profile of the three F4 variants to SDS-PAGEseparated intestinal brush border membrane proteins of pigs differing in: (1) the F4ac-specific antibody response upon oral immunization, (2) the in vitro adhesion of F4+ E. coli to villi, and (3) in their MUC4 genotype. As such we identified six groups of pigs: one negative for all tests and five F4R+ groups. Only two out of five were positive in all three assays and showed the same F4-specific binding bands. Although, putative F4Rs must be present in the other three F4R+ groups, no potential F4-binding band could be identified in those groups. In the last study, the F4-specific binding bands were purified using different methods including an F4acR pull-down assay, sequential chromatographies, continuous-elution electrophoresis followed by separation with 1D or 2D SDS-PAGE and by shotgun mass spectrometry. Several identified candidates were tested for their presence in F4R+ brush border membranes. One of the candidates showed high potential to be an F4acR. Exploring the genetic and biochemical nature of the F4Rs shows to be complex. Till now, F4Rs have been studied for more than 20 years. Yet both the causal mutation and the identity of the F4Rs are still unresolved. Several reasons impede its discovery, including the complexity of the susceptibility phenotypes (due to the three F4 variants, the use of different pig breeds and non-uniform selection of animals in different studies), the complexity of F4R characteristics and limited knowledge of genetic information. Further study on our F4acR candidate will be useful to identify mutation(s) responsible for F4+ ETEC susceptibility.
Please use this url to cite or link to this publication:
author
promoter
UGent and UGent
organization
year
type
dissertation
publication status
published
subject
keyword
pigs, ELISPOT, F4 ETEC, F4R glycoprotein
pages
158 pages
publisher
Ghent University. Faculty of Veterinary Medicine
place of publication
Merelbeke, Belgium
defense location
Merelbeke : Faculteit Diergeneeskunde (kliniekauditorium B)
defense date
2015-11-19 17:00
language
English
UGent publication?
yes
classification
D1
id
6988409
handle
http://hdl.handle.net/1854/LU-6988409
date created
2015-11-23 10:53:26
date last changed
2017-01-16 10:49:53
@phdthesis{6988409,
  abstract     = {Post-weaning diarrhea is mainly caused by F4+ enterotoxigenic Escherichia coli (F4+ ETEC), leading to severe economic losses. Three different F4 variants exist, F4ab, F4ac and F4ad, with pigs showing adhesion of one or more of these variants or completely F4 receptor negative (F4R-). For long-term management of the infection, selection for F4R- pigs and/or vaccination of F4R+ pigs seems to be the best strategies to prevent postweaning diarrhoea. However, neither the causal mutation resulting in F4+ ETEC resistance nor the biochemical nature of the F4R have been determined yet. This thesis aimed to characterize and identify F4R glycoprotein(s).
In the first study, we evaluated the prevalence of the F4+ ETEC susceptible phenotypes among Flemish pig breeds using an in vitro villous adhesion assay. Based on adhesion of the 3 F4 variants, seven different F4+ ETEC susceptible phenotypes were found. Overall, F4ab and F4ac E. coli adhered more numerous to intestinal villi than F4ad E. coli. The completely resistant phenotype E was found in 24.08\% of the pigs. F4acR+ pigs can be orally immunized with F4ac fimbriae, one of the criteria used to select pigs for F4R glycoprotein characterisation. However, most pigs have F4ac-specific maternal antibodies, which mask the serum antibody response upon oral immunization.
Therefore, we established in the second study an ELIspot test to detect the antibody response in piglets having colostral F4ac-specific serum antibodies. This test allowed us to identify circulating F4ac-specific antibody secreting cells (ASCs). Total F4ac-specific ASCs as well as IgA F4ac-specific ASCs revealed an F4ac-specific antibody response in the orally immunized animals with maternal antibodies in the absence of an increase in serum antibodies. Interestingly, the IgA F4ac-specific ASC response on enriched IgA+ B cells elucidated a more robust IgA booster response upon oral immunization of pigs with than without maternal antibodies.
To select potential intestinal F4R glycoproteins, we determined in the third study the binding profile of the three F4 variants to SDS-PAGEseparated intestinal brush border membrane proteins of pigs differing in: (1) the F4ac-specific antibody response upon oral immunization, (2) the in vitro adhesion of F4+ E. coli to villi, and (3) in their MUC4 genotype. As such we identified six groups of pigs: one negative for all tests and five F4R+ groups. Only two out of five were positive in all three assays and showed the same F4-specific binding bands. Although, putative F4Rs must be present in the other three F4R+ groups, no potential F4-binding band could be identified in those groups.
In the last study, the F4-specific binding bands were purified using different methods including an F4acR pull-down assay, sequential chromatographies, continuous-elution electrophoresis followed by separation with 1D or 2D SDS-PAGE and by shotgun mass spectrometry. Several identified candidates were tested for their presence in F4R+ brush border membranes. One of the candidates showed high potential to be an F4acR.
Exploring the genetic and biochemical nature of the F4Rs shows to be complex. Till now, F4Rs have been studied for more than 20 years. Yet both the causal mutation and the identity of the F4Rs are still unresolved. Several reasons impede its discovery, including the complexity of the susceptibility phenotypes (due to the three F4 variants, the use of different pig breeds and non-uniform selection of animals in different studies), the complexity of F4R characteristics and limited knowledge of genetic information. Further study on our F4acR candidate will be useful to identify mutation(s) responsible for F4+ ETEC susceptibility.},
  author       = {Nguyen Van, Ut},
  keyword      = {pigs,ELISPOT,F4 ETEC,F4R glycoprotein},
  language     = {eng},
  pages        = {158},
  publisher    = {Ghent University. Faculty of Veterinary Medicine},
  school       = {Ghent University},
  title        = {The complexity of the small intestinal F4 Escherichia coli receptors in pigs and its role in immunization},
  year         = {2015},
}

Chicago
Nguyen Van, Ut. 2015. “The Complexity of the Small Intestinal F4 Escherichia Coli Receptors in Pigs and Its Role in Immunization”. Merelbeke, Belgium: Ghent University. Faculty of Veterinary Medicine.
APA
Nguyen Van, U. (2015). The complexity of the small intestinal F4 Escherichia coli receptors in pigs and its role in immunization. Ghent University. Faculty of Veterinary Medicine, Merelbeke, Belgium.
Vancouver
1.
Nguyen Van U. The complexity of the small intestinal F4 Escherichia coli receptors in pigs and its role in immunization. [Merelbeke, Belgium]: Ghent University. Faculty of Veterinary Medicine; 2015.
MLA
Nguyen Van, Ut. “The Complexity of the Small Intestinal F4 Escherichia Coli Receptors in Pigs and Its Role in Immunization.” 2015 : n. pag. Print.