Improvement of adjuvant systems to obtain a cost-effective production of high levels of specific IgY.

Incomplete Freund's adjuvant (IFA) is used as standard adjuvant for the production of specific antibodies. In this study, we evaluated the ability of supplementation of IFA with 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] or C-phosphate-guanosine-oligodeoxynucleotide (CpG-ODN) to enhance the quantity of specific IgY found in the eggs of hyperimmunized laying hens. In this comparative study, the fimbrial adhesin F4 of porcine enterotoxigenic Escherichia coli was used as prototype immunogen. Hens of 3 groups received by i.m. injection 20 microg of purified F4 adhesin emulsified with 1 of the following adjuvants: 0.5 mL of IFA alone (F4-IFA group), 0.5 mL of IFA supplemented with 285.6 ng of 1alpha,25(OH)(2)D(3) (F4-IFA-D(3) group), or 0.5 mL of IFA supplemented with 10 microg of CpG-ODN (F4-IFA-CpG group). Hens of 2 control groups received PBS or purified F4 alone. Immunization was repeated after 2 and 5 or 7 wk. Eggs were collected at 3- to 4-d intervals from preimmunization to d 79, and whole eggs were tested to measure the quantity of anti-F4 IgY by a standardized indirect ELISA. The quantity of specific anti-F4 IgY present in eggs from immunized hens of the F4-IFA group increased from d 13 to 79, corresponding to the end of the experiment. The values for this group at each time were considered as 100%. Results obtained for the other adjuvants were expressed in relation to this reference method. Supplementation of IFA with 1alpha,25(OH)(2)D(3) did not result in any enhancement of the quantity of anti-F4 IgY present in the eggs. On the other hand, supplementation of IFA with CpG-ODN resulted in an enhancement of yield up to 942% of the F4-specific antibody response. Moreover, the use of CpG-ODN is a cost-effective and ethical refinement for the production of specific antibodies, permitting a reduction in the number of immunizations needed. In conclusion, this study provides strong evidence for the use of IFA supplemented with CpG-ODN rather than IFA alone for the production of high levels of specific antibody in laying hens.