Diss. (sammanfattning) Göteborg : Göteborgs universistet, 2009. / Härtill 4 uppsatser.

Master of Science in Biomedical Sciences / Department of Diagnostic Medicine/Pathobiology / Weiping Zhang / Heat-labile enterotoxin (LT) is one of the most important toxins produced by enterotoxigenic Escherichia coli (ETEC). It consists of one A subunit (LTA) for intracellular enzymatic activity and five B subunits (LTB) forming a pentamer for binding to host cell receptors. In the last few decades, LT has been extensively studied as a strong immune stimulator, as well as an effective adjuvant with multiple immunomodulatory properties. To understand better the features of LT, we mapped B-cell linear epitopes of the enzymatic A subunit and explored the relationship between these epitopes and the toxicity of LT. Eleven B-cell linear (continuous) epitopes were in silico identified based on online software. In part one of the study, all 11 epitopes were fused into a modified ovalbumin carrier protein respectively. Each recombinant fusion protein was expressed and purified, and was characterized in ELISA and Western Blot using the anti-LT serum. Moreover, each fusion protein was used to immunize mice to determine immune response specific to LT in vivo. A total of eleven epitopes were identified from the LTA subunit. Results showed that anti-LT serum recognized all 11 epitopes, while the mouse immunization study indicated that antibodies derived from epitope 7 (₁₀₅SPHPYEQEVSA₁₁₅) had significantly greater anti-LT antibody titers and neutralized LT enterotoxicity more efficiently than the other epitopes. In part two of the study, to test whether individual epitope plays a role in LT toxicity, 10 epitopes in the A1 domain of LTA subunit were replaced by a foreign peptide respectively and the mutant LTs were examined for enterotoxicity. Data indicated that all these LT mutants showed enterotoxicity abolished. However, these LT mutants formed holotoxin structure and bound to GM1 in vitro. Results from this study indicated that replacement of these LT epitopes did not affect the forming of LT holotoxin structure and the binding to host receptors, indicating LT can serve as a safe vaccine platform to carry foreign antigens. With the immunodominant epitope 7 being kept while other LTA epitopes replaced by epitopes from other ETEC virulence factors, this platform can be used to construct broadly protective multivalent mucosal vaccines against ETEC, and perhaps as a universal platform for vaccines against other enteric diseases.

Thesis (M.P.H.)--University of Texas Health Science Center at Houston, School of Public Health, 2008. / Source: Masters Abstracts International, Volume: 46-06, page: 3219. Adviser: Herbert L. DuPont. Includes bibliographical references

The weaning transition period in pigs is linked to increased vulnerability to enteric disorders, which is partly attributed to destabilisation of the gut microbiota. Post-weaning colibacillosis is an economically important disease of the small intestine, which is most commonly caused by enterotoxigenic Escherichia coli (ETEC) strains. This disease has been variably linked to a diarrhoeal phenotype and decreased growth rate under clinical or sub-clinical conditions, and has been associated with shifts in particular bacterial populations using culturing methods. The emergence of next-generation sequencing technologies such as 16S rRNA gene metabarcoding now allows higher resolution study of complex microbial communities, without being reliant on the ability to culture fastidious micro-organisms. As part of this project, a 16S rRNA gene metabarcoding method was developed and validated to allow qualitative and quantitative measurement of gut microbiota shifts. A series of experimental ETEC challenge trials were carried out to monitor temporal faecal microbiota dynamics (Chapter 2), to further understand ETEC adhesion and shedding dynamics (Chapter 3) and to study potential changes in both ileal and faecal microbiota populations in response to dietary protein manipulation (Chapter 4). The effects of experimental treatments on pig health and performance were also measured as part of each experiment. Temporal shifts in ileal and faecal microbiota structure and stability were observed over the post-weaning period, as well as shifts in relative abundances of particular bacterial phylotypes (P < 0.05) (Chapter’s 2 and 4). ETEC challenge had no effects on faecal microbiota composition, pig health and performance when comparing to samples obtained from sham-challenged pigs (P > 0.05). However, when taking ETEC shedding level into account, variations in both microbiota structure and stability were observed at specific time points (P < 0.05) (Chapter 2). After a single-dose ETEC challenge, ETEC adhesion in the ileum and faecal shedding were evident up to 4 and 6 days post-challenge, respectively (Chapter 3). Changes in ileal microbiota structure and stability were observed in response to ETEC challenge (P < 0.05), with no changes exerted at faecal level (P > 0.05). Additionally, different dietary protein levels were linked to changes in ileal microbiota structure, stability and phylotype relative abundances (P < 0.05). Interestingly, significant differences in ileal microbiota structure were evident in samples obtained from ETEC-challenged pigs fed the low and high protein diets, with the pigs fed the high protein diet having significantly less stable ileal communities at population level (P < 0.05) (Chapter 4). The treatments had no effect on host performance (P > 0.05), but faecal consistency scores were higher in pigs fed the high protein diet (P < 0.05). In conclusion, both ETEC challenge and manipulation of dietary protein level had profound effects on ileal microbiota composition and faecal microbial communities were variable according to ETEC shedding status. These findings have implications for the development of alternative management strategies for enteric diseases in weaner pigs.

L’objectif de cette étude est d’évaluer si trois souches de levure Saccharomycescerevisiae (Lv01, Lv02, Lv03) exercent in vitro des propriétés immuno-modulatrices suite àl’exposition de cellules épithéliales intestinales porcines à Escherichia coli entérotoxinogène(ETEC). Ensuite, nous avons évalué si ces propriétés sont associées à une protection des porceletscontre une infection colibacillaire au post-sevrage et à une stimulation de l’immunité maternellepassive chez la truie.In vitro, nous avons montré que Lv01 exerce une activité anti-inflammatoire suite àl’exposition de cellules épithéliales intestinales porcines à ETEC. Cette activité réside dans 1) lasécrétion des facteurs solubles et est associée 2) à la diminution de la phosphorylation des protéinesERK1/2, p38 et 3) à l’agglutination des ETEC par Lv01. Cependant, Lv01 ne prévient pasl’altération de la barrière épithéliale induite par ETEC.In vivo, à la différence d’expérimentations antérieures, les levures Lv01 et Lv02 neprotègent pas les porcelets contre une colibacillose expérimentale au post-sevrage. Par ailleurs, lestrois souches de levure stimulent l’accroissement des IgG et IgA dans le colostrum et le lait destruies suggérant la transmission d’une meilleure protection immunitaire aux porcelets. / The objective of this work is to investigate whether three Saccharomyces cerevisiaeyeast strains (Lv01, Lv02, Lv03) induce in vitro immuno-modulatory properties in pig intestinalepithelial cells exposed to enterotoxigenic Escherichia coli (ETEC). Thereafter, we assessedwhether these properties are associated to a piglet protection against post-weaning colibacillosisand to a stimulation of maternal passive immunity in sows.In vitro, we have shown that Lv01 induces an anti-inflammatory activity in pig intestinalepithelial cells exposed to ETEC. This activity is induced by 1) secreted soluble factors and isassociated with 2) a decrease of both ERK1/2 and p38 phosphorylation and 3) an agglutination ofETEC by Lv01. However, Lv01 does not prevent the alteration of epithelial barrier integrityinduced by ETEC.In vivo, we have shown that in contrast to previous data, Lv01 and Lv02 do not protectweaned piglets against an experimental ETEC infection. Elsewhere, the three yeast strainsstimulate the immunoglobulin levels (IgG, IgA) in colostrum and milk of sows suggesting thetransfer of a better immune protection to piglets.

<p>Enterotoxigenic Escherichia coli (ETEC), the leading cause of traveler's diarrhea and childhood mortality due to diarrhea in the developing world, has been shown to secrete heat-labile enterotoxin (LT) in association with outer membrane vesicles. However, studies on the effect of LT have been performed using soluble LT, which is not its physiologically relevant presentation context. The effect of LT associated with vesicles and its trafficking within human intestinal epithelial cells were compared with soluble LT. Cytokine responses and trafficking of standardized samples of soluble LT and vesicle-associated LT were evaluated in polarized intestinal epithelial cells. Using real-time PCR, immunoblotting, and ELISAs, we found that compared to soluble LT, vesicle-bound LT showed delayed kinetics in the activation of LT. Vesicles containing LT or not also produced cytokines through different signaling pathways than soluble LT. We found that this difference in signaling was due to different trafficking within the cell. Interestingly, not all LT associated with vesicles is active within cells. Vesicle-associated LT must bind to the host receptor GM1 in lipid rafts to be active within cells. This suggests that although vesicles can deliver large amounts of LT to a cell, much of the LT would be inactive and not produce a physiological response. To test this hypothesis, we attempted to develop animal models for ETEC-induced diarrhea. Although the models were largely unsuccessful, the mouse model appears promising for determining the physiological response of a host to LT as fluid accumulation was observed in response to vesicles containing LT. The results in this thesis provide further understanding of the mechanism of LT-induced diarrhea and emphasize the importance of study toxins in their natural context.</p> / Dissertation

F4 positive enterotoxigenic Escherichia coli (ETEC) is the most common pathogen causing neonatal diarrhea in piglets. The pathogenesis requires the attachment of ETEC to the intestinal brush border, mediated by F4 fimbria. Colostral anti-F4 antibodies and some non-immunoglobulin porcine skim milk proteins can bind F4 and prevent colonization and infection by F4-positive ETEC. Little is known, however, about the F4-binding ability of porcine milk fat globule membrane (MFGM) proteins. In addition, the knowledge of the absorption of porcine colostral proteins into the blood of neonatal piglets is limited, despite the well accepted concept that in neonatal piglets, protein absorption from the intestine is non-selective.<p> In this study, the ability of porcine MFGM proteins to bind purified F4ac (one of the three subtypes of F4 fimbriae) was investigated. Porcine MFGM proteins were first separated by 2D SDS-PAGE and subsequently identified by mass spectrometry. Overlay western Blot was then employed to demonstrate the interaction between porcine MFGM proteins and purified F4ac. Several proteins from porcine MFGM reacted with F4ac, and of these, lactadherin, butyrophilin, adipophilin, and acyl-CoA synthetase 3 reacted strongly. The biological function of these proteins in vivo was not investigated but it is possible that their interaction with F4ac positive ETEC interferes with bacterial attachment and colonization. In order to investigate protein absorption by neonatal piglets after natural suckling, the protein profiles of the plasma of pre-suckling and 24 h post-suckling neonatal piglets were studied by 2D SDS-PAGE. Those plasma proteins that increased prominently after suckling were then identified by mass spectrometry. Only immunoglobulins were unequivocally determined to be absorbed, because they were absent before suckling and present in large quantity in plasma 24 h after suckling. The absorption of other colostral proteins was either equivocal or not detectable by our detection methods. These results suggest that, unlike immunoglobulins, major non-immunoglobulin proteins in porcine colostrum may not be absorbed into systemic circulation in substantial amounts.

F4 positive enterotoxigenic Escherichia coli (ETEC) is the most common pathogen causing neonatal diarrhea in piglets. The pathogenesis requires the attachment of ETEC to the intestinal brush border, mediated by F4 fimbria. Colostral anti-F4 antibodies and some non-immunoglobulin porcine skim milk proteins can bind F4 and prevent colonization and infection by F4-positive ETEC. Little is known, however, about the F4-binding ability of porcine milk fat globule membrane (MFGM) proteins. In addition, the knowledge of the absorption of porcine colostral proteins into the blood of neonatal piglets is limited, despite the well accepted concept that in neonatal piglets, protein absorption from the intestine is non-selective.<p> In this study, the ability of porcine MFGM proteins to bind purified F4ac (one of the three subtypes of F4 fimbriae) was investigated. Porcine MFGM proteins were first separated by 2D SDS-PAGE and subsequently identified by mass spectrometry. Overlay western Blot was then employed to demonstrate the interaction between porcine MFGM proteins and purified F4ac. Several proteins from porcine MFGM reacted with F4ac, and of these, lactadherin, butyrophilin, adipophilin, and acyl-CoA synthetase 3 reacted strongly. The biological function of these proteins in vivo was not investigated but it is possible that their interaction with F4ac positive ETEC interferes with bacterial attachment and colonization. In order to investigate protein absorption by neonatal piglets after natural suckling, the protein profiles of the plasma of pre-suckling and 24 h post-suckling neonatal piglets were studied by 2D SDS-PAGE. Those plasma proteins that increased prominently after suckling were then identified by mass spectrometry. Only immunoglobulins were unequivocally determined to be absorbed, because they were absent before suckling and present in large quantity in plasma 24 h after suckling. The absorption of other colostral proteins was either equivocal or not detectable by our detection methods. These results suggest that, unlike immunoglobulins, major non-immunoglobulin proteins in porcine colostrum may not be absorbed into systemic circulation in substantial amounts.

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