Servizio Comunitario di Informazione in materia di Ricerca e Sviluppo - CORDIS

Immunotherapy using specific egg yolk antibodies in pigs

The objectives of this section were:

- To raise specific antibodies against AEEC virulence factors in hens (egg yolks are a rich source of immunoglubulins that can easily be incorporated into animal or human diets)

- To examine the ability of these antibodies to prevent the development of intestinal colonisation and diarrhoea.

It has clearly been demonstrated in the literature that the intimin Eae and the secreted proteins EspA and EspB are virulence factors that play an important role in the pathogenesis of various AEEC infections. These different virulence factors were thus considered as good candidates for protective immunity.

Primer pairs specific for each of the virulence factors were chosen to amplify by PCR the eae, espA and espB genes from the genomic DNA of enteropathogenic E. coli porcine strain 1390. The different DNA amplified fragments were inserted in the pQE30 expression vector which links a His6 tag at the N-terminal end of the proteins. After transformation in the M15(pREP4) strain, the fusion proteins His-Eae, His-EspA and His-EspB were detected by Western blot analysis revealed with anti-histidine antibodies and with antibodies specifically directed against each of the virulence factors. All fusion proteins were then produced on a large scale and purified on a nickel affinity column in sufficient quantities for immunization of chickens and for use as antigens in the ELISA.

Chickens were immunized with each of the purified proteins. Eggs were collected and egg yolks containing antibodies (IgY) were mixed with PBS, chloroform was added and the overall mixture vortexed. After centrifugation, the water phase containing IgY was conserved. Western blot analysis showed that purified IgY recognized homologous fusion proteins. The titer of the specific IgY for each protein, using homologous purified proteins as antigens in an ELISA, was 1/4800, 1/4000 and 1/5600 for anti-Eae, anti-EspA and anti-EspB IgY, respectively.

AEEC strains originating from the rabbit, pig, dog, bovine, and human (including non-O157 EHEC and EPEC), and producing Eae of the a, b, d, or e subtypes, induced A/E lesions equally well on newborn pig ileal explants. On the other hand, O157:H7 AEEC strains producing Eae of the g subtype adhered to a much lesser extent to the ileal epithelium. However, replacement of the Eae of the g subtype by the Eae of the a-subtype in an O157:H7 strain resulted in induction of A/E lesions to a similar extent as observed for the homologous porcine AEEC strain. These results suggest that Eae of the g subtype of O157:H7 strains recognizes receptors on porcine ileal epithelial cells less well than the Eae of the other known subtypes. Nevertheless, our results confirm that pig ileal explants are an appropriate model for the examination of the effect of specific antibodies on the formation of A/E lesions by AEEC of diverse origin.

Antibodies specific for the mature and carboxy terminal of Eae, Tir and, in the case of the homologous porcine strain 1390, Paa, significantly blocked the development of A/E lesions in ileal explants, both for the homologous strain 1390 and all of the tested AEEC strains from the calf, and human, the latter including both EPEC and O157:H7 EHEC strains. The anti-EspA, anti-EspB, and anti-EspD antibodies did not affect the development of A/E lesions by any of the tested AEEC strains, with the exception of a human EPEC strain, for which the anti-EspA antibodies did significantly block the development of A/E lesions. Anti-Paa antibodies did not block the development of A/E lesions due to this human EPEC strain that produced Eae but not Paa. Hence, the anti-Eae mature, anti-Tir, and anti-Paa antibodies, and possibly the anti-EspA antibodies, were considered as potential candidates for the blocking of development of A/E lesions in vivo.

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