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β-glucans exert receptor-mediated immunomodulating activities, including oxidative burst activity and cytokine secretion. The role of the β-glucan receptors dectin-1 and complement receptor 3 (CR3) in the response of immune cells towards β-glucans is still unresolved. Dectin-1 is considered as the main β-glucan receptor in mice, while recent studies in man show that CR3 is more important in β-glucan-mediated responses. This incited us to elucidate which receptor contributes to the response of innate immune cells towards particulate β-glucans in pigs as the latter might serve as a better model for man. Our results show an important role of CR3 in β-glucan recognition, as blocking this receptor strongly reduced the phagocytosis of β-glucans and the β-glucan-induced ROS production by porcine neutrophils. Conversely, dectin-1 does not seem to play a major role in β-glucan recognition in neutrophils. However, recognition of β-glucans appeared cell type-specific as both dectin-1 and CR3 are involved in the β-glucan-mediated responses in pig macrophages. Moreover, CR3 signalling through focal adhesion kinase (FAK) was indispensable for β-glucan-mediated ROS production and cytokine production in neutrophils and macrophages, while the Syk-dependent pathway was only partly involved in these responses. We may conclude that CR3 plays a cardinal role in β-glucan signalling in porcine neutrophils, while macrophages use a more diverse receptor array to detect and respond towards β-glucans. Nonetheless, FAK acts as a master switch that regulates β-glucan-mediated responses in neutrophils as well as macrophages.

␤-Glucans are well known for their immunomodulatory capacities in humans and mice. For this reason, together with the European ban on growth-promoting antibiotics, ␤-glucans are intensively used in pig feed. However, as shown in the present study, there is much variation in the stimulatory capacities of ␤-glucans from different sources. Since dendritic cells (DCs) are the first cells that are encountered after an antigen is taken up by the intestinal epithelial cell barrier, we decided to investigate the effect of two concentrations (5 and 10 g/ml) of five commercial ␤-glucan preparations, differing in structure and source, on porcine monocytederived dendritic cells (MoDCs). Although all ␤-glucans gave rise to a significant reduction of the phagocytic activity of DCs, only Macrogard induced a significant phenotypic maturation. In addition to Macrogard, zymosan, another ␤-glucan derived from Saccharomyces cerevisiae, and curdlan also significantly improved the T-cell-stimulatory capacity of MoDCs. Most interesting, however, is the cytokine secretion profile of curdlanstimulated MoDCs, since only curdlan induced significant higher expression levels of interleukin-1␤ (IL-1␤), IL-6, IL-10, and IL-12/IL-23p40. Since the cytokine profile of DCs influences the outcome of the ensuing immune response and thus may prove valuable in intestinal immunity, a careful choice is necessary when ␤-glucans are used as dietary supplement.

One of the problems hindering the development of DNA vaccines is the relatively low immunogenicity often seen in humans and large animals compared to that in mice. In the present study, we tried to enhance the immunogenicity of a pcDNA1/faeG19 DNA vaccine in pigs by optimizing the FaeG expression plasmid and by coadministration of the plasmid vectors encoding the A and B subunits of the Escherichia coli thermolabile enterotoxin (LT). The insertion of a Kozak sequence and optimization of vector (cellular localization and expression) and both vector and codon usage were all shown to enhance in vitro FaeG expression compared to that of pcDNA1/faeG19. Subsequently, pcDNA1/faeG19 and the vector-optimized and the vector-codon-optimized construct were tested for their immunogenicity in pigs. In line with the in vitro results, antibody responses were better induced with increasing expression. The LT vectors additionally enhanced the antibody response, although not significantly, and were necessary to induce an F4-specific cellular response. These vectors were also added because LT has been described to direct the systemic response towards a mucosal immunoglobulin A (IgA) response in mice. Here, however, the intradermal FaeG DNA prime-oral F4 boost immunization resulted in a mainly systemic IgG response, with only a marginal but significant reduction in F4 ؉ E. coli fecal excretion when the piglets were primed with pWRGFaeGopt and pWRGFaeGopt with the LT vectors.Intestinal infections with enterotoxigenic Escherichia coli (ETEC) remain problematic for humans, pigs, and calves. The bacteria possess adhesins which allow them to colonize the small intestine and to produce enterotoxins which act locally on enterocytes. These adhesins bind to specific receptors on the enterocyte brush borders, and the absence of these receptors renders the animal resistant to bacterial colonization and consequently to ETEC-induced diarrhea (27). In pigs, F4 (K88) fimbriae are the best-characterized adhesins (37). They are long proteinaceous appendages composed mainly of several hundreds of identical adhesive subunits called FaeG (22).Our laboratory developed a challenge model in pigs in which new vaccination strategies against ETEC can be tested. With this model, it was demonstrated that the oral administration of F4 fimbriae to weaned F4R ϩ pigs led to protection against a subsequent F4 ϩ ETEC infection (35). In practice, a protective mucosal immune response often needs to be induced in the presence of maternal antibodies.Several studies have shown that DNA vaccines, in contrast to conventional vaccines, can successfully prime immune responses in the presence of maternal antibodies, pointing out their potential use in young animals (3). Our previous pcDNA1/faeG19 DNA vaccine, however, was only marginally immunogenic in pigs (42). This is in agreement with other studies in large animals and humans, suggesting poor immunogenicity of DNA vaccines in these species (20,39). Several approaches have been explored to enhance the potency of DNA vaccin...

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