Edith Stuyven

The effect of β-glucans on porcine leukocytes.

beta-Glucans are conserved glucose polymers found in the cell walls of plants, fungi, yeasts and bacteria. They have the capacity to activate innate immunity, thereby enhancing defence barriers. Besides differences in type of linkage and branching, beta-glucans can vary in solubility, molecular mass, tertiary structure, polymer charge and solution conformation. All these characteristics may influence their immunomodulating effects. In this study, the effect of seven beta-glucans that differed in origin (fungi, yeast, seaweed, bacteria or algae) and structure (linear or branched; soluble, gel or particulate) were tested on peripheral blood mononuclear cells (PBMC) and neutrophils of the pig. We looked at lymphocyte proliferation, reactive oxygen species (ROS), production by neutrophils and monocytes and cytokine production. The soluble beta-glucans Laminarin and Sleroglucan did not activate ROS-production of monocytes and neutrophils while the particulate beta-glucans (beta-glucan from algae (Euglena gracilis)) and glucan preparations from bakers yeast (Macrogard, Saccharomyces cerevisiae and Zymosan) had a stimulating effect. The highest stimulation of lymphocyte proliferation occurred by Curdlan (bacteria), Zymosan and the beta-glucan of E. gracilis, especially at high concentrations (200 microg/ml and 800 microg/ml). TNF-alpha was particularly stimulated by Macrogard and S. cerevisiae, while all beta-glucans (except Laminarin) induced IL-1beta. Furthermore, it was interesting that all beta-glucans and in particular Curdlan, gave rise to IL-10 secretion, whereas any beta-glucan induced the release of IL-8, IL-4, IL-12, IL-6 or IFN-gamma.

Enterotoxigenic Escherichia coli (K88) induce proinflammatory responses in porcine intestinal epithelial cells.

Infections with F4(+) enterotoxigenic Escherichia coli (ETEC) causes severe diarrhoea in piglets, resulting in morbidity and mortality. F4 fimbriae are the key virulence factors mediating 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 adhesion to porcine IEC and the secretion of IL-6 and IL-8 by IEC. Remarkably, this cytokine secretion was not abrogated following stimulation with an F4-negative strain. Since this 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 is the dominant virulence factor involved in the induction of proinflammatory responses in IEC. These results indicate a potential mucosal adjuvant capacity of ETEC-derived flagellin and may improve rational vaccine design against F4(+) ETEC infections.

Effect of β-glucans on an ETEC infection in piglets.

The effect of orally administered beta-glucans in protecting pigs against an ETEC infection after weaning was analysed in this study. Three beta-glucans that differed in origin (Saccharomyces cerevisiae (MCG (Macrogard) and G2) or Sclerotium rolfsii (G3)) and/or extraction procedure were tested. Pigs fed for 2 weeks after weaning with these glucans were less susceptible to an F4+ ETEC infection in comparison with the control group. This was evidenced by a reduction in the faecal excretion of F4+ Escherichia coli as well as a reduced F4-specific serum antibody response. This decrease in faecal excretion was statistically significant for pigs fed with the MCG glucan in a first experiment and with the G3 glucan in a second experiment; diarrhoea was milder in the glucan-supplemented groups and was significantly reduced in the MCG-supplemented group. Furthermore, a lower amount of F4-specific IgM antibody-secreting cells (ASC) was found in the lymphoid tissues of pigs fed with G2 or G3 glucans in comparison with the control pig, as well as lower F4-specific IgA ASC in G3-fed pigs in comparison with the control pig. This study showed that beta-glucans can protect against an ETEC infection. Both MCG from S. cerevisiae and G3 from S. rolfsii, resulted in significant effects. To our knowledge, this is the first in vivo study, in which the use of beta-glucans as feed ingredient for just-weaned piglets was tested for their protective effects against ETEC infection.

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

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.

Protection of pigs against Chlamydia trachomatis challenge by administration of a MOMP-based DNA vaccine in the vaginal mucosa

Plasmid DNA (pWRG7079::MOMP) expressing the major outer membrane protein of a human Chlamydia trachomatis serovar E strain was tested for the ability to induce an immune response and protect against experimental genital infection with the same serovar. The vaccine was tested in pigs, as they are genetically and physiologically related to humans and suitable for studying C. trachomatis infection of the genital system. To increase the immune response, GM-CSF, LTA and B and CpG motives were used as adjuvants. GM-CSF was administered seven days before immunization, while the other adjuvants were administered together with the vaccine. Ten pigs were randomly divided into two groups. One group received an intravaginal primo-vaccination and a booster of 500 μg pWRG7079::MOMP, while the other group received the placebo vaccine pWRG7079. All animals were challenged with 10(8) TCID(50) of C. trachomatis serovar E. Pigs immunized with the DNA vaccine showed significantly less macroscopic lesions, vaginal excretion and chlamydial replication in the genital tract, as compared to placebo-vaccinated controls. However, infection could not be completely cleared.

Identification of the porcine C-type lectin dectin-1

Beta-glucans are conserved glucose polymers found in the cell walls of plants, fungi, yeasts and bacteria. They have a backbone of beta-(1-3)-linked glucose units with beta-(1-6)-glucan-linkages. Although a number of receptors are thought to play a role in mediating the biological response to beta-glucans, dectin-1, a C-type lectin, was described as the most important receptor. Dectin-1 belongs to the large family of pattern recognition receptors (PRRs) which recognize conserved pathogen-associated molecular patterns (PAMPs). Here, we report the identification and characterization of dectin-1 in the pig. We identified two major isoforms (GenBank acc. no. FJ386383 and FJ386384), which differ by the presence of a stalk region separating the carbohydrate recognition domain (CRD) from the transmembrane region. Furthermore, a third minor isoform (acc. no. FJ386385) was identified which was a variation of the primary isoform with a deletion in the transmembrane and stalk region. At the nucleotide level, the full length porcine dectin-1 comprises 744bp and is 88% identical to the bovine dectin-1 and 82% identical to the human dectin-1. Messenger RNA transcripts for porcine dectin-1 were detected in the stomach, the small intestine, colon and rectum, the spleen, the mesenterial lymph nodes and the lungs. The transcript was not expressed in the liver, kidneys, the bladder, the heart, the brains and the skin.

Validation of the Chlamydia trachomatis genital challenge pig model for testing recombinant protein vaccines.

Chlamydia trachomatis is a Gram-negative obligate intracellular bacterial pathogen that is the leading cause of bacterial sexually transmitted disease in humans in developing countries. A vaccination programme is considered to be the best approach to reduce the prevalence of C. trachomatis infections. However, there are still no commercial C. trachomatis vaccines. In order to develop effective C. trachomatis vaccines, it is important to identify those antigens that elicit a protective immune response, and to develop new and adequate methods and adjuvants for effective vaccine delivery, as conventional methods have failed to induce protective immunity. In order to test different vaccine candidates, animal models are needed. Former studies have used non-primate monkeys, mice or guinea pig infection models. The present study used a pig model for testing recombinant protein vaccines. Two recombinant proteins, polymorphic membrane protein G (PmpG), and secretion and cellular translocation protein C (SctC), were tested for their ability to create protection in a pig C. trachomatis challenge model. The vaccines were administered subcutaneously with GNE adjuvant. Six weeks later, animals were challenged intravaginally with C. trachomatis serovar E. After a further 4 weeks, the pigs were euthanized. PmpG-immunized pigs were better protected than pigs immunized with the less promising SctC candidate vaccine antigen. Interestingly, significant protection was apparently not correlated with a strong humoral immune response upon subcutaneous immunization. In conclusion, the pig model is useful for studying the efficacy of vaccine candidates against genital human C. trachomatis infection.

Oral Administration of β-1,3/1,6-Glucan to Dogs Temporally Changes Total and Antigen-Specific IgA and IgM

ABSTRACT The effect of oral administration of β-1,3/1,6-glucans from Saccharomyces cerevisiae on humoral immunity in domestic dogs is not known. In this study, 15 beagle dogs were orally given MacroGard tablets, which contain 150 mg of this β-glucan, daily for 4 weeks. At the end of this period, the total serum immunoglobulin A (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 titers 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 1 week after the cessation of the supplementation. In conclusion, the results showed a temporary change in the isotype profile during glucan supplementation.

Oral administration of beta-1,3/1,6-glucan Macrogard fails to enhance the mucosal immune response following oral F4 fimbrial immunisation in gnotobiotic pigs

In this study gnotobiotic animals were used to see if the F4 fimbrial antigen of F4ac+ Escherichia coli is as immunogenic as in conventional pigs. In addition, the adjuvant effect of beta-1,3/1,6-glucans was analysed for the first time in germ-free domestic animals. Oral F4 immunisation of F4 receptor positive (F4R(+)) piglets was able to induce a systemic and mucosal immune response as conventional pigs, although less pronounced. Nevertheless, an IgA response was observed in Peyers patches and mesenteric lymph nodes. Moreover, a dose dependent effect was observed. Oral administration of beta-glucans for 5 weeks was not able to enhance this F4-specific immune response. The only effect of the beta-glucan treatment we observed was a significantly higher IL-1 alpha mRNA expression in the spleen. No significant changes in total serum antibody concentrations and in the volume of the ileal Peyers patches were seen.

Development of a method for isolating bovine colostrum mononuclear leukocytes for phenotyping and functional studies

The present study reports a method for isolating bovine colostrum mononuclear cells (CMC) for phenotyping and functional studies. As well as being an important source of immunoglobulins, colostrum also contains leukocytes that may be of greater importance for passive immunity than has previously been thought. Different protocols have been reported for isolating leukocytes from bovine colostrum, although none of these have been validated, and phenotypic analysis of cell populations has not always been performed. In this study, bovine CMC were isolated by density gradient centrifugation. Cell populations were identified by flow cytometry using antibodies against selected bovine cell surface markers and the proliferative capacity of these cells was determined using a (3)H-thymidine proliferation assay. The mean cell count of isolated CMC was 3 × 10(4) and 1 × 10(5) per mL colostrum for the samples used in the flow cytometric assay and the proliferation assay, respectively. A mean of 25.4 ± 17.1% CMC were identified as T lymphocytes, 2.9 ± 3.0% as B lymphocytes and 32.7 ± 13.7% as macrophages. In terms of proliferation, the mean counts per minute were 4.3 × 10(3) and 1.8 × 10(4) for cells cultured in medium only or in the presence of concanavalin A, respectively, showing that CMC are viable and capable of responding to mitogen stimulation. Isolation of CMC and the subsequent phenotypic analysis of the different subpopulations were repeatable, with agreement indices varying between 0.5 and 1.0. Agreement indices for the proliferation assay were estimated at 0.8.