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Currently, no vaccines are available for edema disease and post-weaning diarrhoea (PWD) in pigs. In the present study, a subunit vaccine containing the F18 fimbrial adhesin FedF was studied. Hereto, recombinant FedF was produced as a fusion protein with maltose-binding protein. Even though the produced MBPFedF was shown to attach in vitro to enterocytes, almost no FedF-specific immune response could be detected after oral administration to piglets. The delivery of FedF to the intestinal mucosa was improved by conjugating the MBPFedF to F4 fimbriae. Indeed, this conjugation induced a systemic and local FedF-specific immune response and led to a reduction in excretion after infection with F18+ E. coli. Although complete protection was not observed, the conjugation between FedF and F4 fimbriae can be considered as a first step towards the development of a combined vaccine against F4+ and F18+ E. coli infections.

A recently developed oral immunisation model in pigs in which F4 (K88) fimbriae of enterotoxigenic Escherichia coli are administered to induce a protective intestinal immunity, was used to determine the optimal inductive sites of the F4-specific intestinal immune response. Hereto, pigs were immunised with F4 orally, in the lumen of the mid-jejunum, ileum or mid-colon. Throughout the small intestine, the highest number of ASC was found following jejunal immunisation, followed by ileal, oral and colonic immunisation. To determine the signifance of Peyer's patches in the induced immune response, F4 was injected into the jejunal Peyer's patches (JPP), lamina propria (LP) and ileal Peyer's patches (IPP). Immunisation in the JPP induced the highest number ASC in the small intestine, whereas immunisation in the LP and IPP resulted in lower intestinal antibody responses. In conclusion, we have shown that the JPP are the major inductive sites of the F4-specific intestinal antibody response. This knowledge could be important when using the pig as an animal model for vaccination studies.

Early vaccination is necessary to protect pigs against postweaning diarrhoea caused by enterotoxigenic Escherichia coli (ETEC). However, at present no commercial vaccine allows successful vaccination. This is partly due to the presence of maternally derived antibodies. Since DNA vaccines are suggested to be superior to protein vaccines in young animals with maternal antibodies, we determined whether the fimbrial adhesin (FaeG) of F4ac(+) ETEC could be used as a plasmid DNA vaccine to prime piglets in a heterologous prime-boost approach. Hereto, pcDNA1/faeG19 was constructed and expression of rFaeG in Cos-7 cells was demonstrated. Thereafter, pigs were immunised (days 0, 21 and 42) intramuscularly by injection or intradermally by gene gun and humoral and cellular immune responses were analysed. Even though responses were low, results demonstrated that intramuscular injection was superior to gene gun delivery for priming the humoral immune response since higher antibody titres were raised, whereas gene gun delivery better induced a cellular response, evaluated by a lymphocyte proliferation assay. Effective priming of the humoral immune response was evidenced by high IgG titres 1 week after a protein boost with purified F4. The low responses to the pcDNA1/faeG19 DNA vaccination suggest that delivery of the DNA and/or the expression of the faeG gene should be improved.

We have used FaeG DNA to immunise piglets by the intradermal (ID) and the intramuscular (IM) route in a heterologous prime/boost model. ID immunisation with DNA resulted in a better induction of cellular immunity, whereas only the IM immunisation could prime an F4(K88)-specific serum IgA response. However, ID administration of plasmid-encoded GM-CSF 1 week before the ID immunisation enhanced the F4-specific humoral and cellular immune response and even primed the F4-specific IgA response more efficiently than the IM immunisation did.

In this study it was analysed whether intramuscular (IM) immunisation of piglets with F4 during the suckling period could protect against oral challenge with F4 + - Escherichia coli and whether addition of 1α,25(OH) 2 D 3 or CpG-ODN could improve this protection. F4-seronegative F4-receptor positive pigs were divided into four groups of five pigs each. The pigs were intramuscularly injected with F4 fimbriae only or supplemented with 1α,25(OH) 2 D 3 (D 3 -group) or CpG-ODN (CpG-group). The control group received PBS in IFA. Seven days after the second immunisation, all pigs were intragastrically inoculated with 1×10 10 CFU of F4 + - E. coli . All F4-injected groups, showed a reduced faecal excretion of F4 + - E. coli . However, this reduction was only statistically significant in the D 3 -group 2 days post challenge. Pigs in the latter group showed a secondary antibody response upon challenge, indicating that F4-primed memory B-cells were present in the gut-associated lymphoid tissues at that moment. CpG-ODN, on the other hand, did not enhance the F4-specific antibody response. However, CpG-ODN significantly increased the F4-specific as well as mitogen-induced proliferation of pheripheral blood monomorphonuclear cells indicating a direct or indirect overall effect on T-lymphocytes. In conclusion, supplementation with 1α,25(OH) 2 D 3 or CpG-ODN improved protection against an F4 + - E. coli infection. This protection was most obvious for 1α,25(OH) 2 D 3 and indicates its potential use in veterinary vaccines against enteropathogens.

To develop a vaccine against Escherichia coli -induced post-weaning diarrhea and edema disease, insights in the induction of the protective immune response following infection with these pathogenic E. coli is needed. Therefore, the fimbriae-specific antibody response of newly weaned pigs following infection with the Shiga-like toxin type II variant (SLT-IIv) producing F18 + verotoxigenic E. coli (VTEC) (strain 107/86) was compared with the response following an infection with LT producing F4 + enterotoxigenic E. coli (ETEC) (strain GIS 26). F4 + ETEC were able to colonize the gut very soon after infection, as peak excretion of F4 + E. coli bacteria was seen 2 days post-infection (dpi), but had already disappeared 7 dpi. On the other hand, F18 + VTEC infection resulted in a slower colonization of the gut as the peak excretion of F18 + E. coli was observed between 3 and 5 dpi, but this colonization remained longer as F18 + E. coli were detected till 9 dpi in feces. Furthermore, this fast colonization pattern of F4 + ETEC is accompanied with the presence of F4-specific antibodies in mucosal tissues and serum from 4 dpi onward, with maximal amounts of F4-specific IgA in the jejunal lamina propria and serum 7 dpi. In contrast, F18-specific IgA was only readily detected in the jejunal lamina propria 15 dpi and showed a maximum serum titer 21 dpi. Besides this faster induction and higher antibody response, the switch from IgM to IgA and IgG was also earlier following the F4 + ETEC infection.