Colidiarrhea and colienterotoxemia caused by F4+ and/or F18+ enterotoxigenic E. coli (ETEC) strains are the most prevalent infections of suckling and weaned pigs. Here we tested the immunogenicity and protective effectiveness of attenuated F18ac+ non-ETEC vaccine candidate strain against challenge infection with F4ac+ ETEC strain by quantitative phenotypic analysis of small intestinal leukocyte subsets in weaned pigs.We also evaluated levamisole as an immune response modifier (IRM) and its adjuvanticity when given in the combination with the experimental vaccine. The pigs were parenterally immunized with either levamisole (at days -2, -1 and 0) or with levamisole and perorally given F18ac+ non-ETEC strain (at day 0), and challenged with F4ac+ ETEC strain 7 days later.At day 13 the pigs were euthanatized and sampled for immunohistological/histomorphometrical analyses. Lymphoid CD3+, CD45RA+, CD45RC+, CD21+, IgA+ and myeloid SWC3+ cell subsets were identified in jejunal and ileal epithelium, lamina propria and Peyer’s patches using the avidin-biotin complex method, and their numbers were determined by computer-assisted histomorphometry. Quantitative immunophenotypic analyses showed that levamisole treated pigs had highly increased numbers of jejunal CD3+, CD45RC+ and SWC3+ cells (p<0.05) as compared to those recorded in nontreated control pigs.In the ileum of these pigs we have recorded that only CD21+ cells were significantly increased (p<0.01). The pigs that were treated with levamisole adjuvanted experimental vaccine had significantly increased numbers of all tested cell subsets in both segments of the small intestine. It was concluded that levamisole adjuvanted F18ac+ non-ETEC vaccine was a requirement for the elicitation of protective gut immunity in this model; nonspecific immunization with levamisole was less effective, but confirmed its potential as an IRM.

Enterotoxigenic Escherichia coli (ETEC) infection is the most common type of porcine postweaning colibacillosis (PWC). Among fimbriae of porcine ETEC strains the best studied family of fimbriae are the members of F4 adhesins, existing in at least three variants: ab, ac, ad. Active immunization against porcine PWC is difficult due to: i) ETEC strains are only one of the essential predisposing factors, ii) the success of vaccinal antigen uptake depends on the presence of enterocyte receptors for F4 adhesins, iii) the intestinal immune system may react with tolerance or hypersensitivity to the same antigens depending on the dose and form of the vaccinal immunogen, and iv) kinetics of the specific immune responses may be different in the case of F4 (earlier) and the other ETEC adhesins, particularly F18 (later). The aim of this study was to test the effectiveness of a live attenuated F4ac+ non-ETEC vaccine against porcine PWC by analyzing quantitative differences in the small intestinal lymphoid and myeloid cell subsets of immunized (with or without levamisole given as an adjuvant) vs control non-immunized pigs. Four week-old pigs were intragastrically immunized with a vaccine candidate F4ac + non-ETEC strain 2407 at day 0, challenged 7 days later with a virulent F4ac+ strain ETEC 11-800/1/94, euthanatized at day 13 and sampled for immunohistology. Non-immunized pigs received saline at day 0 and were processed as the principals. Immunophenotypes of lymphoid and myeloid cell subsets were demonstrated within jejunal and ileal mucosa by immunohistochemical avidinbiotin complex method and corresponding morphometric data were analyzed using software program Lucia G for digital image analyses. Monoclonal antibodies reactive with surface molecules on porcine immune cells such as CD3, CD45RA, CD45RC, CD21 and SWC3 enabled clear insight into distribution patterns and amount of these cells within the gut-associated lymphoid tissues (GALT) examined. The numbers of jejunal and ileal cell subsets tested were significantly increased (at P<0.5 or lower) in both principal groups (vaccinated or levamisole primed-vaccinated) of pigs, compared to those recorded in the control non-vaccinated pigs. Based on the histomorphometric quantification of porcine intestinal immune cells from the GALT compartments tested, it is possible to differentiate the responses of pigs immunized by an experimental mucosal vaccine from those of non-immunized pigs.

Immunohistological identification/localization of immunomodulating neuropeptides [vasoactive intestinal polypeptide (VIP) and substance P (SP)] and enzyme nitric oxide synthase (NOS) as well as histomorphometric analyses of kinetics of their release and development of respective nerve fibers density during postnatal ontogenesis of porcine intestinal mucosal immune system (IMIS), were performed in order to assess the role of these molecules involved in maturation of the IMIS. The kinetcs of reactions to VIP, SP and NOS were demonstrated in the samples of jejunum and ileum from conventionally reared pigs. The samples were obtained at 0, 7, 14, 21, 28, 35, 42 and 49 days of age and processed for immunohistological staining. The VIP+ reaction was prevalently visible in the epithelial layer, lamina propria and Lieberkühn crypts (Lc) but also in the submucosa and lamina muscularis along blood and lymphatic vessels. The SP+ fibers were regularly distributed along enteric neurons in the muscular layer. The reaction to NOS was demonstrated in both mucosa and submucosa of ileum and jejunum and in the ileal Peyer's patches (PP). Intensity of the reaction was more pronounced in the epithelial layer and numerous NOS+ cells were observed around the Lc and inside the follicles of the PP. Also, we have noticed NOS+ blood vessels, particular neurons and nerve fibers in the submucosa and muscular layer of the small intestine. By analyzing quantitative patterns of SP+, VIP+ fibers and release of NOS we have concluded that intensity of their reactions gradually increases with age, except a short period of stagnation after weaning (at age of 28 days), reaching the highest values in the pigs aged between 42 and 49 days. The values obtained by Sperman rank order correlation test (rs) between days of age of pigs and intensity of the reactions in their jejunum/ileum to VIP (rs=0.97/0.95), SP (rs=0.97/0.97) and NOS (rs=0.98/0.95), respectively, showed positive correlations (P<0.05) according to Roemer Orphal scale. Current study showed that post-natal development of porcine IMIS was accompanied by a substantial increase in the secretion of neuropeptides/enzyme tested and that these molecules may participate in the functional maturation of immunoregulatory/bactericidal mechanisms of the local (intestinal) immune defense in young pigs.

Levamisole (2, 3, 5, 6-tetrahydro-6-phenylimidazole 2,1-b thiazole) is a well-known nonspecific stimulator of host defence mechanisms. In previous investigations, we have found that levamisole acts on cell-mediated immunity in challenge-induced porcine postweaning colibacillosis (PWC). We assume that levamisole could also act synergistically on humoural immune response when applied as an adjuvant with vaccine candidate strains for oral immunization of weaned pigs against PWC. The influence of levamisole in combination with experimental F4ac(+) nonenterotoxigenic Escherichia coli (non-ETEC) vacinal strain on proliferation of IgA(+) cells was examined in 4-week-old weaned pigs experimentally infected with ETEC. We have performed identification and morphometric quantification of the plasma cell phenotype within jejunal/ileal mucosa. Plasma cells were identified by immunohistochemistry with monoclonal anti-IgA antibodies and quantifying by use of digital image analysis. Quantification of IgA(+) cells from levamisole-primed vaccinated and challenge-infected weaned pigs showed significantly increased number (P < 0.05 for both jejunum and ileum) compared with those observed in unprimed vaccinated/challenge-infected controls. It is suggested from these results that levamisole may contribute in initiation of local humoural immune response to enteric pathogens, such as enterotoxigenic E. coli.

Colidiarrhea and colienterotoxemia caused by F4+ and/or F18 + enterotoxigenic E. coli (ETEC) strains are the most prevalent infections of suckling and weaned pigs. Here we tested the immunogenicity and protective effectiveness of attenuated F18ac + non-ETEC vaccine candidate strain against challenge infection with F4ac + ETEC strain by quantitative phenotypic analysis of small intestinal leukocyte subsets in weaned pigs.We also evaluated levamisole as an immune response modifier (IRM) and its adjuvanticity when given in the combination with the experimental vaccine. The pigs were parenterally immunized with either levamisole (at days -2, -1 and 0) or with levamisole and perorally given F18ac + and myeloid SWC3 + cell subsets were identified in jejunal and ileal epithelium, lamina propria and Peyer's patches using the avidin-biotin complex method, and their numbers were determined by computer-assisted histomorphometry. Quantitative immunophenotypic analyses showed that levamisole treated pigs had highly increased numbers of jejunal CD3 + , CD45RC + and SWC3 + cells (p<0.05) as compared to those recorded in nontreated control pigs.In the ileum of these pigs we have recorded that only CD21 + cells were significantly increased (p<0.01). The pigs that were treated with levamisole adjuvanted experimental vaccine had significantly increased numbers of all tested cell subsets in both segments of the small intestine. It was concluded that levamisole adjuvanted F18ac + non-ETEC vaccine was a requirement for the elicitation of protective gut immunity in this model; nonspecific immunization with levamisole was less effective, but confirmed its potential as an IRM.