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

Abstract

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.

Levamisole stimulates proliferation of circulating and intestinal immune cell subsets, gut health and performance in weaned pigs

“…The results obtained on the effectiveness of LEVA used as an immunomodulator to: (1) enhance proliferation of peripheral blood and intestinal immune cell subsets, (2) maintain gut health, and (3) improve productivity and performance of 4-wk-old weaned pigs based on the study during period of 5 wk postweaning were generally in agreement with many of our previous observations (Lackovic´et al 1997;Bozˇic´et al 2002aBozˇic´et al , b, 2003Bozˇic´et al , 2006Bozˇic´et al , 2011Janjatovic´et al 2008;Kovsˇca Janjatovic´et al 2010) and by others (Reyero et al 1979;Hennessy et al 1987;Kumar et al 1999;Bilandzˇic´et al 2010a, b). However, the study design and duration of our current research were rather different (i.e., immunization with a single dose of LEVA only, oral route of its application, age of pigs and considerably longer observation period), included measurements of more parameters (i.e., nutritional, bacteriological and immunological) than in similar studies Kovsˇca Janjatovic´et al 2009) and conveyed the pioneer data on influence of the drug on distribution and frequency patterns of naı¨ve lymphoid cells residing ileal mucosa of 9-wk-old pigs.…”

“…More recently, we have observed that the drug, when given either alone or in combination with live oral F18ac ' non-ETEC vaccine, stimulated proliferation of the ileal CD21 ' B cells in 6-wk-old pigs (Kovsˇca Janjatovic´et al 2009). Also, the LEVA-adjuvanted live oral F4ac ' non-ETEC vaccine enhanced recruitment of CD45RA ' naı¨ve and CD45RC ' memory lymphoid cells, SWC3 ' macrophages and CD21 ' B cells residing in ileal mucosa of weaned pigs more strongly than did the vaccine alone (Kovsˇca Janjatovic´et al 2010). Pigs weaned at less than 3 wk of age have lower lymphocyte proliferative responses than pigs weaned at an greater age (Blecha et al 1983) and may have a reduced growth rate as a result of reduced immune protection (Bailey et al 1993).…”

Increased number of intestinal villous M cells in levamisole - pretreated weaned pigs experimentally infected with F4ac+ enterotoxigenic Escherichia coli strain

“…Recently, we have reported, based on the histomorphometric quantification of porcine intestinal immune cells, that it is possible to differentiate the responses of pigs immunized with levamisole-adjuvanted experimental mucosal vaccines against PWC from those of nonimmunized pigs. 10 , 11 Based upon these observations, we highlighted the query of whether and how the already recognized immunogenicity/adjuvanticity of levamisole 8 , 9 can influence de novo differentiation of the antigen-sampling M cells and consequently provide more feasible and effective immunization with mucosally delivered vaccines against enteric pathogens, such as porcine ETEC. In the case of positive answer, the study on development of an effective M cell-targeted vaccine against porcine PWC should be adjuvanted with levamisole in order to circumvent weak or tolerogenic responses.…”

“…The F4ac + E. coli strains used for either immunization or challenge infection were described in a previous study of our group. 11 Mouse IgG1 (clone C-04) monoclonal antibody (mAb), reactive with porcine cytokeratin peptide 18 16 was used as primary antibody (Biovendor Laboratory Medicine, Heildelberg, Germany). Polyclonal F(ab') 2 rabbit anti mouse IgG:HRP antibody (AbD Serotec, Oxford, UK) was used as secondary antibody.…”

“… 8 , 9 The attempts to validate the influence of levamisole on the intestinal cellular immunity against PWC, when applied alone or in the combination with live oral F4ac + or F18ac + vaccines, confirmed its potentials as an immunomodulator and mucosal adjuvant. 10 , 11 However, our knowledge about mechanisms operating at the mucosal surfaces of the small intestine following oral immunization of pigs with a live vaccine candidate F4ac + non-ETEC strain is still limited. Further research should include antigen sampling microfold (M) cells providing transcytotic antigen delivery through the follicle-associated epithelium (FAE) to the underlying immune cells within the gut associated lymphoid tissues (GALT), such as ileal Peyer's patches.…”