The use of a double antibody sandwich ELISA and monoclonal antibodies for the assessment of porcine IgM, IgG and IgA concentrations.

Double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) have been developed for the assessment of IgM, IgG and IgA concentrations in porcine serum. Isotype-specific monoclonal antibodies (mAb) were used for coating and detection. The DAS-ELISAs were examined for their ability to detect each isotype and for the assay variations. A computer programme was used to verify the parallelism of the slope of each serum sample with the slope of standard/reference serum, a prerequisite for reliable estimation of Ig concentrations. The DAS-ELISAs are easy to perform and highly specific, have adequate detection levels (ranging from 7 to 50 ng ml-1) and are very reproducible, as illustrated by the inter- and intra-assay variation coefficients (ranging between 4.9 and 7%). To illustrate the applicability of the ELISAs we assessed Ig concentrations in pig sera sampled from birth to young adulthood.

Antigen presenting cells and B-cells in the pig.

The central position of antigen presenting cells (APC) in the immune system and the heterogeneity of the APC family are discussed; both aspects are illustrated with data from species other than the pig. Thereafter the limited work on porcine APC is reviewed. The section on B-cells, the effector cells of the humoral immune system, exclusively focuses on 'porcine data'.

Conversion of orally induced suppression of the mucosal immune response to ovalbumin into stimulation by conjugating ovalbumin to cholera toxin or its B subunit.

Oral pretreatment of mice with ovalbumin (OVA) not only suppressed a subsequently induced systemic immune response ('oral tolerance') but also suppressed, even more effectively, a subsequently induced intestinal IgA response. In contrast, pretreatment with OVA conjugated to cholera toxin (CT) or its B subunit (CTB) resulted in a stimulative effect. The stimulative effect was enhanced when unconjugated OVA and polymerized OVA were removed from the OVA-CT (B) conjugate mixtures by affinity chromatography. Thus, the effect of oral pretreatment depends on the balance between tolerizing and stimulating components in the conjugate mixture. As OVA-CTB conjugates were at least as effective as OVA-CT conjugates in stimulation of the intestinal immune response, we concluded that the ability of the OVA conjugates to bind to the intestinal mucosa is a prerequisite in inducing the stimulative effect. These observations further demonstrate that conjugation of a protein antigen to an appropriate carrier can convert the nature of the immunization from suppressive into stimulative.

Apathogenic, intestinal, segmented, filamentous bacteria stimulate the mucosal immune system of mice.

Segmented filamentous bacteria (SFBs) are apathogenic autochthonous bacteria in the murine small intestine that preferentially attach to Peyer's patch epithelium. SFBs have never been cultured in vitro. We have studied the effects of SFBs on the immune system of the host. Mice monoassociated with SFBs were compared with germ-free mice and with mice without SFBs but with a specific-pathogen-free (SPF) gut flora. SFBs versus no microbial flora raised the number of lymphoid cells in the lamina propria of the ileal and cecal mucosa, raised the number of immunoglobulin A (IgA)-secreting cells in the intestinal mucosa, produced elevated IgA titers in serum and intestinal secretions, and enhanced the concanavalin A-induced proliferative responses of mesenteric lymph node cells. The SPF flora had effects similar to but less pronounced than those mediated by SFBs. The results indicate that SFBs stimulate the mucosal immune system to a greater extent than do other autochthonous gut bacteria.

Comparison of an indirect haemagglutination assay and an ELISA for diagnosing Fasciola hepatica in experimentally and naturally infected sheep.

An enzyme-linked immunosorbent assay (ELISA) with somatic (S) or excretory-secretory antigens (ES) was compared with an indirect haemagglutination assay (IHA) for ability to detect antibodies against Fasciola hepatica in sheep. The specificity of both assays was determined by testing sera collected from sheep experimentally or naturally mono-infected with Fasciola hepatica, Haemonchus contortus, Ostertagia circumcincta, Cooperia curticei, Taenia ovis, Eimeria spp., Trichostrongylus vitrinus, Trichostrongylus colubriformis or Nematodirus battus respectively. With S or ES antigens the specificity of the ELISA was 98% or 95% respectively, whereas the specificity of the IHA was 86%. Antibodies directed against Fasciola hepatica were detected by the ELISA with S or ES antigens from 2 weeks after infection until the end of the experiment, whereas the IHA detected antibodies from week 3. We conclude that the ELISA with S antigens compares favourably with the IHA and can be used for the serodiagnosis of ovine fasciolosis in the Netherlands.

Manipulation of intestinal immune responses against ovalbumin by cholera toxin and its B subunit in mice.

We studied the effect of mucosal presentation of ovalbumin (OVA) conjugated to cholera toxin (CT) or cholera toxin B subunit (CTB) on the intestinal immune responses against OVA. Mice were primed intraperitoneally (i.p.) with OVA in a water-in-oil emulsion and boosted intraduodenally (i.d.) with OVA conjugated to CT or CTB in various molar ratios. Responses were evaluated by testing intestinal secretions for OVA-specific antibodies and by quantifying the OVA-specific antibody secreting cells (ASC) in the lamina propria of the small intestine. OVA-CT conjugates were tested in a molar ratio ranging from 1.8:1 to 4500:1. OVA-CTB conjugates were tested in a molar ratio ranging from 0.25:1 to 500:1. The optimum intestinal immune response was reached at a molar ratio of 1.8:1 for OVA-CT and 5:1 for OVA-CTB. The binding capacity of OVA-CTB, but not of OVA-CT, to GM1 ganglioside corresponded with the capacity to enhance the intestinal immune response. The effect of conjugating CTB or CT to OVA on the immune response against OVA was more striking when mice were not only boosted i.d., but also primed i.d. Both OVA-CT and OVA-CTB induced detectable immune responses, whereas free OVA did not. Therefore, the carrier effect of CT or CTB is essential to trigger a mucosal immune response against OVA when presented mucosally only. We conclude that enhancing antigen uptake greatly facilitates mucosal immune responses.

[Mucosal immunity]. / Mucosale immuniteit.

The mucosal immune system protects against infections that enter the body via the mucosae. This article describes how the mucosal immune system functions and how mucosal immune responses can be induced by vaccination. It also discusses various vaccination regimens that maximize the induction of mucosal immune responses against bacterial and viral infections. One paragraph of the article discusses the possibilities of developing vaccines that induce a mucosal immune response against parasite infections.

Induction of an enteric Ig-response against ovalbumin and stimulation of the response by cholera toxin and its B-subunit in mice.

We induced ovalbumin (OA)-specific antibody responses in the murine small intestine and quantitated the responses by counting ovalbumin-specific antibody-containing cells (OACC) in the intestinal lamina propria, and by measuring anti-OA Ig titers in intestinal secretions. OA is a "weak" mucosal antigen and we could only induce intestinal anti-OA responses by intraperitoneal, primary injection of OA in water-in-oil emulsion and a mucosal booster with OA. Double staining of OACC for OA- and isotype-specificity demonstrated that 95% of all mucosal (intestinal) OACC produced IgA, whereas 95% of all systemic (splenic) OACC produced IgG. The intestinal anti-OA response could be stimulated by addition of cholera toxin (CT) or cholera toxin B-subunit (CTB) during the mucosal booster immunization. The stimulatory effect of CT did not depend on covalent coupling of CT and OA, while the stimulatory effect of CTB required covalent coupling of CTB with OA. Mucosal (intraduodenal) application of OA as such does not prime for a mucosal and systemic OACC response but induces tolerance. We demonstrated that coupling of OA with CT or CTB could overcome the inability of mucosal application of OA to prime for a mucosal OACC response. Although CT proved to be much more effective than CTB in stimulation of mucosal immune responses and possibly in prevention of tolerance induction, our results indicate that CTB is a useful (and safer) alternative.

Background (spontaneous) immunoglobulin production in the murine small intestine before and after weaning.

The ontogeny of the murine intestinal B-cell compartment before and after weaning was studied by quantitative analysis of immunoglobulin-secreting cells (Ig-SC) in the small intestine (SI). Before weaning, few Ig-SC were detected in the SI, whereas spleen and bone marrow already contained many Ig-SC. The number of Ig-SC in the SI started to increase immediately after weaning. Comparing early-weaned mice with non-weaned mice of the same age clearly demonstrated that weaning brought on the development of Ig-SC in the SI. The influence of a gut flora on the number of Ig-SC in the SI was examined by comparing the number of Ig-SC in the SI of conventionally housed, specific pathogen free (SPF) and germ-free mice. A bacterial flora was apparently needed for the normal development of Ig-SC in the SI. Comparing mice containing an aerobic Gram-negative bacterial flora with mice containing only an anaerobic Gram-positive bacterial flora demonstrated that the type of bacterial flora is relatively unimportant. No evidence was found that circulating maternal antibodies suppressed the development of the "spontaneous" intestinal and systemic B cell response. The results show that bacterial colonization of the intestine plays a pivotal role in the development of the Ig-SC compartment in the SI.

Quantification of antigen-specific antibody-secreting cells in the small intestine and other lymphoid organs of mice after oral booster immunization.

The intestinal immune response of mice against ovalbumin (OVA) was quantified by isolating lymphoid cells from the small intestine (SI) and testing them for antigen-specific immunoglobulin (Ig) secretion. The isolation procedure for functionally active lymphoid cells from the SI, originally developed to quantify the number of 'background' Ig-secreting cells in the SI, proved to be a useful method for evaluating antigen-specific intestinal immune responses quantitatively. The method was able to detect antigen-specific antibody-secreting cells (ASC) in the SI even when these cells occurred at a minimum frequency of only 0.006%. When mice were primed intraperitoneally (i.p.) with polymerized OVA and given an oral OVA booster immunization, OVA-specific ASC appeared in the SI from Day 3 after booster. After i.p. priming and an i.p. booster these cells could not be detected in the SI. The OVA-specific IgA-ASC responses in various organs after oral booster immunization were compared. From Day 5 after booster, when the response peaked, most OVA-specific IgA-ASC occurred in the SI. This suggested that these cells are mainly responsible for the OVA-specific antibodies demonstrated by ELISA in intestinal secretions from Day 6 after oral booster immunization. It is concluded that the quantitative method used in this study detects antigen-specific ASC in the SI with great sensitivity and could be used to evaluate immunization regimes aimed at inducing intestinal mucosal immune responses.

Background (spontaneous) immunoglobulin production in the murine small intestine as a function of age.

The development of the intestinal B-cell compartment in C3H/He mice was studied as a function of age by quantification of the number of intestinal immunoglobulin-secreting cells (Ig-SC). Before and at weaning, the number of Ig-SC in the small intestine (SI) was below 10(3) Ig-SC per SI. During the first few weeks after weaning, this number rose steeply and continued to rise until the mice were about 48 weeks old, when a maximum of more than 25 x 10(6) Ig-SC per SI was found. After 1 year of age the number of Ig-SC decreased. At all ages, the great majority of Ig-SC in the SI produced IgA. The increase of the number of IgA-SC in the SI after weaning is reflected in the amount of IgA in intestinal secretions measured by ELISA. The number of Ig-SC in the SI was compared with the number of Ig-SC found in spleen, bone marrow, Peyer's patches and mesenteric lymph nodes. Striking differences were observed between the SI and the other organs tested in total number, isotype distribution and kinetics of the increase of Ig-SC during ontogeny. These differences are discussed in relation to the regulation of the immune response in the SI and the migration patterns of lymphocytes in mucosal tissues.

Contribution of immunoglobulin-secreting cells in the murine small intestine to the total 'background' immunoglobulin production.

In this study we investigated the contribution of the small intestine of C3H/He mice to the spontaneous ('background') immunoglobulin (Ig) production in terms of the number of Ig-secreting cells (Ig-SC), and compared the results with the numbers of Ig-SC found in various lymphoid organs (spleen, bone marrow, mesenteric lymph nodes and Peyer's patches). The results show that in C3H/He mice of 20 weeks of age, on average 16 x 10(6) Ig-SC can be isolated from the small intestine. Almost all of these Ig-SC produce IgA. Compared to the other lymphoid organs, the small intestine contains more than 80% of all Ig-SC present in adult C3H/He mice. These results are in agreement with the need to maintain relatively high levels of Ig at the mucosal surfaces, especially of secretory IgA, for the prevention of penetration of these surfaces by micro-organisms. In man and mouse most of these Ig are supposed to be produced locally in the underlying mucosal tissues and subsequently transported across the epithelium. Although the IgM and IgG levels in serum are predominantly maintained by non-mucosae associated lymphoid organs, the results of this study clearly indicate that the mucosal tissues are the major site of 'background' Ig-production.

Improved procedure for the isolation of functionally active lymphoid cells from the murine intestine.

An isolation procedure for functionally active lamina propria lymphoid cells (LPL) from the murine intestine is described. The procedure involved EDTA-dithiothreitol incubation of intestinal tissue to remove epithelial and intraepithelial cells, followed by collagenase digestion of the basement membrane to liberate part of the LPL. The LPL were suspended by squeezing the remaining tissue strips through a nylon gauze filter. Functional activity was tested by enumeration of the immunoglobulin-secreting cells in the cell suspensions obtained by an isotype-specific protein A plaque-forming cell assay. On average 1-2 X 10(8) LPL were isolated from the intestine of C3H/He mice. 11% of these cells actively secreted Ig. From these Ig-secreting cells 99% produced IgA. The isolation procedure described in this paper permitted a higher recovery of viable cells than has previously been obtained with other methods.

Purification of a heat labile dermonecrotic toxin from culture fluid of Pasteurella multocida.

A highly pure heat-labile dermonecrotic toxin (DNT) of Pasteurella multocida was isolated from bacterium-free broth culture fluid. The protocol for the isolation included the following steps: ammonium sulphate precipitation, gel filtration, ion exchange chromatography and preparative polyacrylamide gel electrophoresis (PAGE). About 1 mg of purified DNT was recovered from 3 l of broth culture fluid. The final product was toxic for embryonic bovine lung (EBL) cells, lethal for mice, dermonecrotic in the guinea pig skin test and inactivated by heating at 56 degrees c. The recovery of biological activity was about 5% that of the original culture fluid and the specific activity had increased about 4000 times. After sodium dodecyl sulphate (SDS)-PAGE and silver staining a single band appeared, indicating that the purified DNT was free from contaminating proteins. The molecular weight of the toxin was approximately 125,000 daltons. The minimal toxic dose of DNT protein for embryonic bovine lung cells was about 2 ng, the minimal dermonecrotic dose in the guinea pig skin test was about 80 ng and the 50% lethal dose for mice about 300 ng.