In this Masters dissertation, various doses of solubulized crude F4 fimbrial protein in conjunction with the adjuvants CpG ODN and porcine â-defensin 1 (pBD-1) were used to enhance the F4-specific intestinal immune response against Enterotoxigenic Escherichia coli (ETEC) F4 in post-weaned pigs. Using the mechanically shearing method we isolated the F4 fimbrial protein of ETEC with a molecular weight of 26 kDa. We verified this using a Western blot probed with a rabbit anti-F4 fimbrial antibody. Binding of the F4 fimbrial protein to the F4 receptor (F4R), present on the brush border of the villi in the small intestine of pigs, was demonstrated using an in vitro villus adhesion assay (IVVA). To demonstrate specificity rabbit polyclonal and mouse monoclonal anti-F4 antibodies, or the F4 protein were used to inhibit the adhesion of ETEC F4ac to F4R positive (F4Rpos) villi. <p>To examine immunogenicity of the 500 micrograms (ìg) of the F4 were administered into surgically created jejunal gut-loops in pigs. Three weeks later Peyers patches (PP) from immunized and control loops as well as gut-wall tissue were analyzed for their F4-specific antibody secreting cells (ASCs) by a modified enzyme linked immunosorbent spot (ELISPOT) assay. The F4-specific immune response in the serum was analyzed by an enzyme linked immunosorbant assay (ELISA). High numbers of F4-specific ASCs were isolated from the loops of pigs that contained high levels of the F4R. Conversely nominal or low numbers of F4-specific ASCs were found in loops of pigs expressing low levels of the F4R or no F4R (F4Rneg). The IVVA was used to categorize the pigs into either F4Rpos or F4Rneg animals. <p>Next three different concentrations of the crude F4 protein 50, 250, and 500 µg in the loops of individual pigs were used to analyze if dose affected the F4-specific immune response. Interestingly dose had no effect on the magnitude of the response. Therefore we hypothesized that the F4-specific immune response in the loops could be enhanced through the use of the adjuvants CpG ODN 2007 and pBD-1. The F4 protein was co-administered with either CpG ODN 2007 or pBD-1 and immune responses were assessed after 3 weeks. However neither CpG ODN 2007 nor PBD-1 at the doses used made an improvement in the immune response. Thus, these results demonstrated that the expression level of the F4R was the most important parameter for eliciting of the local immune response against the F4 protein. Furthermore our studies revealed that both F4Rneg and F4Rpos pigs responded to F4 immunization, however the former respond only nominally to F4-immunization in the loops. Moreover, an inverse relationship existed between the level of the F4-specific IgG in the serum and the F4-specific immune response seen in the loops. Thus our findings have important implications for oral vaccination using fimbrial based antigens (Ags) that utilize a receptor for their immunogenicity. Our results indicate that only animals with high levels of enterocyte F4R will have the ability to elicit high levels of protective F4-specific anti-fimbrial antibodies in their intestine after oral immunization. Therefore unless an effective adjuvant is available, animals with low to moderate levels of the fimbrial receptor in their small intestine will mount only weak immune responses making herd immunity after vaccination currently unattainable.

In this Masters dissertation, various doses of solubulized crude F4 fimbrial protein in conjunction with the adjuvants CpG ODN and porcine â-defensin 1 (pBD-1) were used to enhance the F4-specific intestinal immune response against Enterotoxigenic Escherichia coli (ETEC) F4 in post-weaned pigs. Using the mechanically shearing method we isolated the F4 fimbrial protein of ETEC with a molecular weight of 26 kDa. We verified this using a Western blot probed with a rabbit anti-F4 fimbrial antibody. Binding of the F4 fimbrial protein to the F4 receptor (F4R), present on the brush border of the villi in the small intestine of pigs, was demonstrated using an in vitro villus adhesion assay (IVVA). To demonstrate specificity rabbit polyclonal and mouse monoclonal anti-F4 antibodies, or the F4 protein were used to inhibit the adhesion of ETEC F4ac to F4R positive (F4Rpos) villi. <p>To examine immunogenicity of the 500 micrograms (ìg) of the F4 were administered into surgically created jejunal gut-loops in pigs. Three weeks later Peyers patches (PP) from immunized and control loops as well as gut-wall tissue were analyzed for their F4-specific antibody secreting cells (ASCs) by a modified enzyme linked immunosorbent spot (ELISPOT) assay. The F4-specific immune response in the serum was analyzed by an enzyme linked immunosorbant assay (ELISA). High numbers of F4-specific ASCs were isolated from the loops of pigs that contained high levels of the F4R. Conversely nominal or low numbers of F4-specific ASCs were found in loops of pigs expressing low levels of the F4R or no F4R (F4Rneg). The IVVA was used to categorize the pigs into either F4Rpos or F4Rneg animals. <p>Next three different concentrations of the crude F4 protein 50, 250, and 500 µg in the loops of individual pigs were used to analyze if dose affected the F4-specific immune response. Interestingly dose had no effect on the magnitude of the response. Therefore we hypothesized that the F4-specific immune response in the loops could be enhanced through the use of the adjuvants CpG ODN 2007 and pBD-1. The F4 protein was co-administered with either CpG ODN 2007 or pBD-1 and immune responses were assessed after 3 weeks. However neither CpG ODN 2007 nor PBD-1 at the doses used made an improvement in the immune response. Thus, these results demonstrated that the expression level of the F4R was the most important parameter for eliciting of the local immune response against the F4 protein. Furthermore our studies revealed that both F4Rneg and F4Rpos pigs responded to F4 immunization, however the former respond only nominally to F4-immunization in the loops. Moreover, an inverse relationship existed between the level of the F4-specific IgG in the serum and the F4-specific immune response seen in the loops. Thus our findings have important implications for oral vaccination using fimbrial based antigens (Ags) that utilize a receptor for their immunogenicity. Our results indicate that only animals with high levels of enterocyte F4R will have the ability to elicit high levels of protective F4-specific anti-fimbrial antibodies in their intestine after oral immunization. Therefore unless an effective adjuvant is available, animals with low to moderate levels of the fimbrial receptor in their small intestine will mount only weak immune responses making herd immunity after vaccination currently unattainable.

Diss. Univ. Zürich, 2004. - Ref.: Bruno S. Frey ; Korref.: Simon Gächter.

Diss. Univ. München, 2004.

Nearly all individual members of the animal kingdom have gastrointestinal tracts which feature unique cellular compositions, geometries, and temporal dynamics. These guts are distinct enough from one another, even across siblings or even across the same individual at different points in space and time, that defining meaningful scientific representations of those features is difficult. Studying these guts is also innately challenging as it requires accessing to the insides of the enclosed 3D volumes. The work presented here describes tools and methodologies designed to address these difficulties. To investigate gut motility, we constructed a combined light sheet fluorescence and differential interference contrast microscope to obtain videos of larval zebrafish (Danio rerio) gut motility and to obtain 3D information about nearby fluorescently tagged cells. Using advanced computer vision algorithms, we quantified aspects of zebrafish gut motility which have never before been characterized, then used that information to identify the effects of different genetic, chemical, and physiological states of zebrafish gut motility. Finally, we designed and constructed an instrument for automating 3D microscopy for future studies. This dissertation includes previously published and unpublished co-authored material.

Background: Recent research showing how dietary interventions substantially influence the potential presence of widespread and stable bacterial core phyla in the human colon has garnered a considerable amount of attention. Because the human gut can play a major role in host health, there is currently some interest in observing how diet influences human gut microbial composition and how changes in diet affect the potential for gut microbiota to transform mercury. This study aims to discover how different kinds of diet affect the nature and magnitude of microbial Hg transformations in the human gut environment. Methods: Fecal samples have been collected from 5 human male individuals at University of Ottawa and stored at -80ºC for further investigation. Using high throughput DNA amplicon sequencing targeting the 16s rRNA V4 region, we investigated the microbial community structure of the gut in 5 healthy male. Mercury biotransformations in the pooled fecal sample have been carried out using stable isotopes of mercury (198HgCl2 and Me199HgCl) and analysis was conducted by using inductively coupled plasma mass spectrometry (ICP-MS). Results and conclusions: We were not able to detect any significant Hg methylation or MeHg demethylation. We suspect this is due to Enterobacteria dominating the microbial community structure after 96h; Although Enterobacteria are part of the typical microbiota of a healthy individual, they do not possess genes required for Hg methylation. As such, our microbial data support our chemical analyses. We were not able to identify whether a change in diet affected Hg transformations in the human gut environment.

No description available.

<p> Understanding the mechanisms of microbiome assembly during host development is crucial for successful modulation of the gut microbiome to improve host health and growth. Detailed characterization of the swine gut microbiome through meta-analysis allows us to understand the dynamics of microbial community succession, as well as the transient and natural variations between timepoints and animals. A total of 3,313 fecal samples from over 349 pigs covering 60 time points (from birth to market age) from 14 publications were included in the meta-analysis in Chapter 2. Alpha diversity continuously increased during early stages of animal growth and increased at a slower rate in the following stages. Random forest regression identified 30 OTUs as potential microbiota biomarkers for modeling swine gut microbiome development and the external validation suggested the generalization and benchmarking role of our models in application to future microbiome studies conducted in suckling and weaning pigs. In Chapter 3, a total of 924 fecal samples from 44 newborn piglets over 21 time points (day one of age until 41 days of age) were collected every two days and community composition, assembly, and succession was determined using the V4 region of the 16S rRNA gene. Alpha diversity continuously increased during the suckling stage, yet there was no significant increase in alpha diversity during days post-weaning. Post-weaning in-feed antibiotics consistently decreased the microbial diversity and changed the community structure in both low birth weight (LBW) and normal birth weight (NBW) piglets. Delayed gut microbial community maturation was observed in LBW piglets on post-weaning days compared with NBW. Heterogeneity of the gut microbial community between piglets linearly decreased over time, as revealed by the within-time Bray-Curtis dissimilarities. Intra-individual variance both in community structure and genus abundance indicates the importance of repeated measurements for reliable observations. Dirichlet multinomial mixtures analysis supported an age-dependent microbiome developmental pattern and identified microbial taxa that are age-discriminatory. Our study addresses ecological processes shaping the swine gut microbiome between piglets with contrasting birth weights and receiving post-weaning antibiotics. Persistent gut microbiota immaturity in LBW piglets suggests that efforts to accelerate microbial community succession might improve LBW piglet growth performance and disease resistance. </p>

The absorption and distribution of metals via the gut of fish is not well known. Consequently, the aim of the present study was to describe the movement of metals along the gut, their absorption and binding to gut tissues, and their distribution to the internal tissues following model dietary exposure. Two different approaches were employed, an in vivo gastric dosing procedure, and an in vitro gut bag protocol and two different metals were studied: an essential metal, zinc, and a non-essential (and more toxic) metal, cadmium. The dietary uptake and distribution of zinc and cadmium to 0.3 kg rainbow trout (Oncorhynchus mykiss) was examined at l5°C at 1, 2, 3, or 7 days following a single bolus dose to the stomach of 0.5 mM of radio labelled metaL After exposure, all internal organs and the remaining carcass were individually counted for radioactivity. Uptake, distribution and excretion of both zinc and cadmium was rapid, occurring largely within the first 24 h of exposure. By 24 h, fish exposed to Zn had absorbed 20.0% of the dose, 21.0% was bound in the gastrointestinal tissues and the remainder was either excreted (38.1 %) or was present in the gut lumen (20.9%). Cadmium showed a much different pattern of uptake, with only 2.9% of the dose absorbed after 24 h, and the remainder found either in the gut tissue (30.2%) and the lumen (19.0%) or excreted (47.9%). Over the following six days, very little uptake and internal metal redistribution occurred.When exposed to higher doses of metal in vivo (0.5 - 50 mM), there were distinct differences in the handling of the two metals. Zinc concentrations in the gut tissues continued to rise at higher doses until apparent saturation. In contrast, gut tissues were saturated with cadmium at the lowest dose employed (0.5 mM). Both metals bound most avidly to the distal intestine but all gut tissues had a higher binding capacity for zinc, as compared to cadmium. Target tissues (liver, gills, kidney) all saturated with zinc at high doses. In contrast, cadmium concentrations in these tissues continued to rise in a linear fashion with increasing dose. In vitro studies revealed that the most important region of the gut for metal uptake in rainbow trout was the mid-intestine. Studies using the metabolic uncoupler, 2,4-DNP, suggested that the transfer of both zinc and cadmium across intestinal cells was passive at the brush border membrane, but was at least partly dependent on A TP for movement across the basolateral membrane. Furthermore, this transport mechanism was not shared by calcium, as the presence of calcium had no inhibitory effect on the transport of either metal. Mucus within the intestinal lumen appears to have a higher binding affinity but lower capacity for cadmium than zinc. Calcium did not displace cadmium from the mucus layer. In contrast, zinc was displaced by an equimolar exposure to calcium in the medium. Gut mucus apparently impedes the movement of metals along the intestine with the extent of the delay likely being related to the binding affinity of the metal. The impediment was greatest for cadmium, as 10% of the metal remained in the lumen of fish exposed in vivo, after a period of7 days. In contrast, only 2% ofthe original dose of zinc remained in the gut lumen after only 3 days. / Thesis / Master of Science (MSc)

Cardiovascular disease (CVD) is the leading cause of death in the United States. Recently, the gut microbiota has been implicated in the pathophysiology and progression of CVD. Experimental evidence suggests that high fat feeding alters the functional composition of the gut microbiota (dysbiosis); leading to increased translocation of the pro-inflammatory, endotoxin, and increased production of the pro-atherogenic, trimethylamine-N-oxide (TMAO). Together, these changes are hypothesized to accelerate CVD progression. Conversely, administration of gut microbiota modulating agents, such as antibiotics and probiotics, attenuate high fat feeding induced CVD in rodent models. In humans, the capacity to produce TMAO following L-carnitine or phosphatidylcholine challenges is abolished after receiving broad spectrum antibiotics for a period of one week. However, whether gut modulation over a longer period of time decreases fasting serum endotoxin, fasting plasma TMAO, and CVD risk in response to high fat feeding has been unexplored in humans. To address these issues we conducted a randomized, placebo controlled, parallel group designed, controlled feeding study in healthy, non-obese males receiving the multi-strain probiotic, VSL #3 (or placebo), while a consuming a high fat diet for 4-weeks. First, we tested the hypothesis that VSL #3 would attenuate the rise in serum endotoxin and consequent arterial stiffening following high fat feeding in healthy, non-obese males. Second, we tested the hypothesis that VSL #3 would attenuate the rise in plasma TMAO concentrations following high fat feeding in healthy, non-obese males. In contrast to our first hypotheses, serum endotoxin concentrations and arterial stiffness did not change in response to high fat feeding or with VSL#3 treatment. Interestingly, VSL #3 significantly attenuated the increase in body mass (+ 1.4±0.4 vs. +2.3±0.3 kg; P < 0.05) and fat mass (+0.7±0.1 vs. + 1.4±0.3 kg; P < 0.05) following high fat feeding compared to the placebo. In contrast to our second hypothesis, probiotic supplementation did not attenuate the rise in plasma TMAO following high fat feeding. Future studies are necessary to elucidate the mechanisms responsible for the prevention of body mass and fat mass gain with VSL#3 supplementation following high fat feeding. In addition, studies are needed to determine whether higher doses of VSL #3, other single or multispecies probiotics, prebiotics, or synbiotics attenuate the production of the proatherogenic, TMAO. / Ph. D.