Secretion of immunomodulating neuropeptides (VIP, SP) and nitric oxide synthase in porcine small intestine during postnatal development.

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 regularily 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 postnatal 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.

Histomorphometric evaluation of intestinal cellular immune responses in pigs immunized with live oral F4ac+ non-enterotoxigenic E. coli vaccine against postweaning colibacillosis.

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. Immuno-phenotypes of lymphoid and myeloid cell subsets were demonstrated within jejunal and ileal mucosa by immunohistochemical avidin-biotin 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.