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Protective immune response during pig AEEC infections

The specific objectives are:

- To analyse the immune response, especially cytokine, during pig AEEC infections
- To construct a DNA vaccine specific for AEEC infection
- To analyse the immune response of this DNA vaccine.

Cytokine production during AEEC infection:

Although A/E lesions have been experimentally reproduced in newborn piglets, such lesions have been much more difficult to induce in older conventional pigs. We used two different strategies to analyse the immune response during AEEC infection in pigs.

The first strategy was to use a ligated loop model in pig. Ligated intestinal loops were prepared in 4 weeks old pigs and inoculated with 108 CFU of bacteria. Pigs were evaluated for colonisation and AE lesion using histopatholgy, immuno-histochemistry and cytokine expression level. In each pig there were two loops inoculated with the same strains: AEEC 1390 or D5, (both O45, Intb) or the negative control (123). The results indicated that the colonisation and AE-lesions were 80-90% reproducible. We also analyse the local immune response on the different ileal samples using a RT-PCR technique or a DNA array specific for the pig immune system. This array contains 62 genes including 20 cytokines, 10 chemokines and 12 immunological receptors. We demonstrated that inflammatory cytokine genes are induces by both pathogenic (D5 and 1390) and non-pathogenic (123) strain of E. coli. By contrast the expression of IL-8 is only induced by AEEC strains.

The second strategy was to examine the development of A/E lesions in weaned pigs treated with dexamethasone, an immunosuppressant agent. We demonstrated that dexamethasone, given orally for 7 days at an immunosuppressive dose, enhanced the development of A/E lesions weaned pigs experimentally challenged with the 1390 strain. We successfully reproduce typical intimate adherence leading to A/E lesions. The local intestinal cytokine response following challenge of weaned pigs with the 1390 strain was also investigated. A significant upregulation of IL-1, IL-6, IL-8 and IL-12p40 mRNA were observed in the ileum of control weaned pigs challenged with the 1390 strain and showing few A/E lesions, but not in pigs having received dexamethasone prior to experimental challenge and showing more extensive A/E lesions.

Construction of DNA vaccines:
DNA vaccines are able to induce antigen-specific cellular and humoral immune responses. It is also possible to manipulate the immune response generated through the co-delivery of DNA encoding for cytokines. Wa constructed DNA plasmids encoding for two bacterial antigens (i.e. Eae, and Tir) which are considered as good candidates for protective immunity as well as DNA plasmids encoding for 3 porcine cytokines (GM-CSF, IL-4 and IL-6). The cytokines, already cloned and sequenced in pigs, presented adjuvant properties. GM-CSF is secreted by multiple cells and is able to recruit professional antigen-presenting cells. IL-4 and IL-6 have been implicated in the development of mucosal immunity and in the induction of IgA.

Cytokine and antigen specific DNA fragments were then inserted in the pcDNA3.1/V5-His-TOPO expression vector which links in frame a V5 epitope and a 6 histidine (His6) tag at the N-terminal end of the proteins. Constructs were then verified by a sequence analysis of the entire insert. The gene expression in eukariotic cells was then evaluated by western blot analysis using supernatant of transfected in COS-7 cells.

Immune response upon DNA vaccination:
Two animal experiments were performed in order to analyse the immunogenicity of the DNA vaccine constructions. The first experiment included 4 groups of 3 piglets. Each group was vaccinated 3 times at two weeks intervals with a different plasmid combination (Eae+Tir; Eae+Tir+GM-CSF; Eae+Tir+IL-4; Eae+Tir+IL-6). The different plasmids were injected subcutaneously at the base of the ear and electroporation was used to improve transfection efficacy. In the second experiment, 24 pigs divided in 4 groups were followed up for 7 weeks (between 4-11 weeks of age). The animals were vaccinated 3 times at two weeks intervals with different constructions (unvaccinated control; vaccinated with Eae+Tir; vaccinated with Eae+Tir+IL-4; vaccinated with Eae+Tir+IL-6)

In both experiments, blood samples were taken before the vaccination and bimonthly thereafter. The animals were followed for two month, their humoral immune response was analysed by western blot and ELISA. The first experiment indicates that some animals have antibody directed to intimin before immunisation and that vaccination increased the percentage of positive animals and/or their titre. However, due to the limited number of animal per group and the variability of their response, it was impossible to compare the adjuvant of the three cytokine tested. In the second experiment we failed to see any effect of the vaccination.

Related information

Reported by

INRA- Laboratoire de Pharmacologie-Toxicologie
180 chemin de Tournefeuille
31000 Toulouse
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