Histomorphometric characteristics of immune cells in small intestine of pigs perorally immunized with vaccine candidate F18ac+ nonenterotoxigenic E. coli strain

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.Key words: nonspecific/specific immunization, E. coli, gut immune cells, pigs.Porcine colidiarrhea and colienterotoxemia induced with F4⁺ and/or F18⁺ enterotoxigenic Escherichia coli (ETEC) strains are economically the most significant diseases of swine which account for moderate to high mortality rates and growth retardation, causing death of 5 million pigs per year in the World. Protection from ETEC is a constant challenge due to high genetic flexibility of this widespread bacterial organism.The virulence characteristics of ETEC are strongly dependent on the production of fimbrial adhesins and enterotoxins (Nagy and Fekete, 1999).The ability of adhesion of ETEC to intestinal wall is mainly due to the production of fimbriae. Enterotoxins produced by adherent ETEC strains act locally on enterocytes and stimulate increased water and electrolyte secretion and decreased fluid absorption. Several types of porcine ETEC are known today, including ETEC strain producing F18 fimbriae with their variants “ab” and “ac” (Bretschinger et al., 1990; Nagy and Fekete, 1999; Zang et al., 2007). The ETEC strains causing diarrhea mostly express F4 or F18 adhesins (Fairbrother et al., 2005; Zang et al., 2007). Nearly all known E. coli enterotoxin genes are produced by ETEC strains expressing either F4 or F18 fimbria. Zhang et al. (2007) have conclude that the dominant pathotypes causing diarrhea in weaned pigs are porcine ETEC strains expressing either F4 fimbria and heat-labile (LT) / heat stable (STb) toxins or LT/STb/EAST1 toxins, or F18 fimbria and STa/STb/Stx2e toxins. However, F18ab is more frequently associated with Shiga like toxin 2e, whereas F18ac is more frequently associated with enterotoxin STI (Cheng et al., 2005). Olasz et al. (2005) showed that the 200-kb plasmid, called pF18, contained the genes responsible for F18 fimbriae production.The curing of F18⁺ ETEC strain, i.e. loss of plasmid carrying the heat stable toxin genes in bacterial mutants has been performed by the plasmid transformation and conjugation following co-culturing of donor and recipient strains as reported earlier (Olasz et al., 2005). New vaccination strategies include the oral immunization of pigs with live avirulent nontoxigenic E. coli strains carrying the fimbrial adhesins F4 and/or F18 (Fairbrother et al., 2005).Receptors for F18ab and F18ac variants are increasingly produced up to the weaning age and the fimbriae F18ac seem to have more receptors around the ileal Peyer''s patches (Nagy et al., 1992). The colonization of the small intestine by an F18⁺ ETEC strain causes enterotoxemia. The typical clinical symptoms of the disease are neurological signs such as ataxia, convulsions and paralysis (Vögeli et al., 1996). It is well known that enterotoxic colibacillosis produces significant losses in two different age groups of pigs: first among newborn pigs and later at the postweaning age (Nagy and Fekete, 1999). The disease usually starts a few days after lacteal protection completely ceases (within the first 2 weeks after weaning), especially when weaning occurs at 3–4 weeks of age.Thus, the success of a vaccine against porcine colidiarrhea and colienterotoxemia depends upon applying it in the most efficient form at the optimal time and matching the right protective antigens with the type of virulence factors of ETEC present in the given animal population (Nagy and Fekete, 2005).The gut mucosal immune system contains specialized lymphoid tissues where environmental antigens are presented inducing B- and T-cell responses (Stokes et al., 1994). These responses are regulated by T cells and cytokines and they lead to plasma cell differentiation and the secretion of IgA antibodies onto intestinal mucosal surfaces.The aggregated lymphoid tissue such as Peyer’s patches and solitary lymphoid cells in the lamina propria both play important roles in the induction and regulation of immune responses in the gut associated lymphoid tissues (GALT) (Lacković et al.,1997b). Such organization of the GALT may provide immune protection at mucosal surfaces where the infection actually occurs (McGhee et al., 1992). Bertschinger et al. (2000) demonstrated the protective effects of a live oral vaccine containing F18 fimbria against porcine postweaning diarrhea and oedema disease.In this study we have examined the distribution and quantitative patterns of the subsets of T and B cells as well as of macrophages and secretory IgA⁺ plasma cells within GALT compartments of 4 weeks old pigs perorally immunized with an attenuated F18ac⁺ non-ETEC vaccine candidate strain against porcine colienterotoxemia. Additionally, we have evaluated adjuvanticity of levamisole in the combination with the experimental vaccine and its immunostimulatory effect when applied as an immune response modifier (IRM). Levamisole (2,3,5,6-tetrahydro-6-phenylimidazole thiazole), was originally described as a highly effective anti-helminthic compound (Thienpont et al., 1966). Subsequent studies have established its ability to restore and enhance depressed immune responses in domestic food animals and to act as an effective adjuvant for parenteral and oral vaccines (Mulcahy and Quinn, 1986; Jenkins and Hurdle, 1989; Božić et al., 2002).