MOMP-based DNA vaccination against Chlamydia trachomatis infection in a pig model

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Enterotoxigenic Escherichia coli induce pro-inflammatory responses in porcine intestinal epithelial cells

F4+ enterotoxigenic Escherichia coli (ETEC) cause severe diarrhoea in both neonatal and weaning piglets, resulting in morbidity and mortality. F4 fimbriae are a key virulence factor involved in the attachment of F4+ ETEC to the intestinal epithelium. Intestinal epithelial cells (IEC) are recently being recognized as important regulators of the intestinal immune system through the secretion of cytokines, however, data on how F4+ ETEC affect this cytokine secretion are scarce. By using ETEC strains expressing either polymeric, monomeric or F4 fimbriae with a reduced polymeric stability, we demonstrated that polymeric fimbriae are essential for the adhesion of ETEC to porcine IEC as well as for the secretion of IL-6 and IL-8 by ETEC-stimulated intestinal epithelial cells. Remarkably, this cytokine secretion was not abrogated following stimulation with an F4-negative strain. As this ETEC strain expresses flagellin, TLR5 mediated signalling could be involved. Indeed, porcine IEC express TLR5 and purified flagellin induced IL-6 and IL-8 secretion, indicating that, as for other pathogens, flagellin seems to be the dominant virulence factor involved in the induction of proinflammatory responses in IEC upon ETEC infection. These results indicate a potential mucosal adjuvant capacity of ETEC-derived flagellin and may improve rational vaccine design against F4+ ETEC infections

Oral β-glucans modulate systemic antigen responses in dogs and pigs

The cell wall glucans of yeasts and fungi consist of a linear backbone of -1,3-linked glucosylunits with -1,6-linked side chains (1, 2). Although a lot is already known about the mechanism of action of -1,3/1,6-glucans on the innate immune system (3, 4), there is still a lot to be learned about their effects on the adaptive immune system in mammals. We aimed to determine if oral supplementation could modulate a systemic immune response. The latter was examined in pigs using a model antigen, but also in dogs analyzing the response against a parenteral vaccine. In three experiments using newly weaned pigs, Macrogard, a β-1,3/1,6-glucan from Saccharomyces cerevisiae, was administered in the feed during three different time periods (one, two and three weeks) and the adjuvant effect of this β-glucan was determined on a systemic immunisation with thyroglobulin. A first immunisation occurred during β-glucan supplementation, while the second one occurred after ceasing the administration. Macrogard exerted significantly higher thyroglobulin-specific primary immunoglobulin (Ig) M and secondary IgA antibody responses in serum. However, Macrogard suppressed the thyroglobulin-specific proliferation of peripheral blood mononuclear cells. A higher dose of Macrogard significantly increased thyroglobulin-specific IgM but not IgA responses, and the animals itself showed hyperaemia. Suppression of the T-lymphocyte proliferation might account for the absence of the switch from IgM to IgA. Weight gain and feed conversion were also determined, without significant differences between groups. In another study, also dogs were orally given Macrogard in tablets, daily for four weeks. At the end of this period, the total serum IgA level decreased significantly in the group treated with the glucan compared to that in the control group as well as compared to the concentrations before supplementation. In contrast, the total serum IgM level rose significantly, whereas no effect on the IgG level occurred. Similar changes were seen in Bordetella-specific IgA and IgM titres following vaccination during the supplementation period. The IgA concentration also became significantly lower in the saliva and tears of the glucan group than in the placebo group. The effects disappeared one week after the cessation of the supplementation. There seems to be a temporary decrease in the switch from IgM to IgA due to oral Macrogard supplementation in dogs probably by its suppression of T-lymphocyte proliferation as seen in pigs. In conclusion, oral β-glucans are able to modulate the humoral as well as the cellular immunity against a systemically administered antigen

Oral β-1,3/1,6-glucans as immunmodulators in pigs

The cell wall glucans of yeasts and fungi consist of a linear backbone of -1,3-linked glucosylunits with -1,6-linked side chains (1). Although a lot is already known about the mechanism of action of -1,3/1,6-glucans on the innate immune system (2), there is still a lot to be learned about their effects on the adaptive immune system in mammals. We aimed to determine if oral supplementation could modulate a systemic immune response. The latter was examined in pigs using a model antigen. In three experiments using newly weaned pigs, Macrogard, a β-1,3/1,6-glucan from Saccharomyces cerevisiae, was administered in the feed during three different time periods (one, two and three weeks) and the adjuvant effect of this β-glucan was determined on a systemic immunisation with thyroglobulin. A first immunisation occurred during β-glucan supplementation, while the second one occurred after ceasing the administration. Macrogard exerted significantly higher thyroglobulin-specific primary immunoglobulin (Ig) M and secondary IgA antibody responses in serum. However, Macrogard suppressed the thyroglobulin-specific proliferation of peripheral blood mononuclear cells. A higher dose of Macrogard significantly increased thyroglobulin-specific IgM but not IgA responses, and the animals itself showed hyperaemia. Suppression of the T-lymphocyte proliferation might account for the absence of the switch from IgM to IgA. Weight gain and feed conversion were also determined, without significant differences between groups. In conclusion, oral β-glucans are able to modulate the humoral as well as the cellular immunity against a systemically administered antigen

Intestinal health and mucosal immunity in the young animal

One of the strategies to maintain optimal gut health in animal production in the absence of antibiotic growth promoters is to support the active immunity of the animal. Immunity at the level of the digestive system is concentrated in the mucosal immune system of the gut associated lymphoid tissue (GALT). Therefore, the GALT is the obvious target if we want to increase intestinal immunity. M cells and dendritic cells sample the intestinal contents and bring pathogens in close contact with macrophages, B and T cells in specialized regions in the intestinal epithelium like e.g. the Peyer’s patches (inductive site). Macrophages destroy the pathogen and stimulate the B and T cells to launch an immune response that is specific to this pathogen. Depending on the pathogen and the co-stimulatory signals this response can be the activation of cellular (cytoxic T lymphocytes (CTL)) or humoral (antibodies) immunity. Antibodies (IgA) are secreted at the effector site and transported through the epithelial cells to neutralize the pathogens already in the intestinal lumen. Macrophages thus act at the first line and stimulate a whole cascade of events leading to final antibody production. The intense contact between intestinal contents and immune cells at the mucosa offers opportunities to modulate intestinal immunity via the feed. While immunostimulating activities are claimed on many feed additives like phytoproducts, organic acids, chelates, pre- and probiotics and many others these additives mostly have only bacteriostatic effects or indirect effects on the immune system. On the contrary, it has been widely demonstrated that β-glucans have a direct immunostimulating effect. More specifically they bind to a receptor on macrophages and increase their activity towards stimulation of B and T cells. As a consequence, increased antibody titers after vaccination and better protection after challenges with a pathogen have been observed when β-glucans were incorporated in the feed. However, not all β-glucans are equally effective and their interaction with the receptor on the macrophage depends on their primary and secondary structure (degree of polymerization (DP) and degree of substitution (DS)). Since immunization and challenge trials are laborious, time consuming and therefore very costly we propose to develop a model system based on in vitro assays that can be used to evaluate the immunomodulating property of feed additives. Β-glucans with different purity, DP and DS can thus be compared. Most if not all sources of β-glucans that are used in feed are impure or semi-purified compounds that contain a variety of oligosaccharides including mannan-oligosaccharides or MOS. These molecules can interfere with the attachment of pathogenic bacteria (e.g. enterotoxic Escherichia coli or ETEC) to the intestinal epithelial thus reducing their colonization. Next to immunomodulation this is another aspect of the possible positive effect of these compounds on gut health. In our lab, interference of attachment of bacteria to the intestinal epithelium can also be quantified in vitro. As outlined above, once a pathogen is sampled out of the intestinal contents a local immune response in the intestinal mucosa is generated in different steps. At first macrophages and other phagocytic cells (like dendritic cells ; non-specific immunity) take up the pathogen. They destroy the pathogen with enzymes and O-radicals and process it for presentation to local T cells. Phagocytic cells also secrete pro-inflammatory cytokines that stimulate the activity and proliferation of those T and B cells that are most fit to attack the type of pathogen that was presented by the phagocytic cell (specific immunity). Finally the B cells will produce antibodies (IgA) that can be secreted in the intestinal lumen to neutralize the pathogens. The benefits and values of these assays are that the activity of all the samples within each test will be comparable. Also, the results will give an activity profile of each sample, indicating at what level the immunological response is most probably modulated when the sample is included in the feed. Furthermore, the capacity of a sample to interfere with attachment of E coli is a measure of protection against attachment and colonization of the intestinal epithelium in vivo. And finally, validation of the results of the in vitro screening towards gut health and protection against pathogenic challenges will have to be performed by in vivo tests

The polymeric stability of the Escherichia coli F4 (K88) fimbriae enhances its mucosal immunogenicity following oral immunization

&lt;p&gt;Only a few vaccines are commercially available against intestinal infections since the induction of a protective intestinal immune response is difficult to achieve. For instance, oral administration of most proteins results in oral tolerance instead of an antigen-specific immune response. We have shown before that as a result of oral immunization of piglets with F4 fimbriae purified from pathogenic enterotoxigenic Escherichia coli (ETEC), the fimbriae bind to the F4 receptor (F4R) in the intestine and induce a protective F4-specific immune response. F4 fimbriae are very stable polymeric structures composed of some minor subunits and a major subunit FaeG that is also the fimbrial adhesin. In the present study, the mutagenesis experiments identified FaeG amino acids 97 (N to K) and 201 (I to V) as determinants for F4 polymeric stability. The interaction between the FaeG subunits in mutant F4 fimbriae is reduced but both mutant and wild type fimbriae behaved identically in F4R binding and showed equal stability in the gastro-intestinal lumen. Oral immunization experiments indicated that a higher degree of polymerisation of the fimbriae in the intestine was correlated with a better F4-specific mucosal immunogenicity. These data suggest that the mucosal immunogenicity of soluble virulence factors can be increased by the construction of stable polymeric structures and therefore help in the development of effective mucosal vaccines.&lt;/p&gt;</p