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 1alpha,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 1alpha,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 x 10(10) CFU of F4(+)-E. coli. All F4-injected groups, showed a reduced faecal excretion of W-E. coli. However, this reduction was only statistically significant in the D3-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 1alpha,25(OH)(2)D-3 or CpG-ODN improved protection against an F4+-E. coli infection. This protection was most obvious for 1alpha,25(OH)(2)D-3 and indicates its potential use in veterinary vaccines against enteropathogens. (C) 2002 Elsevier Science Ltd. All rights reserved.