Improving adjuvant systems for polyclonal egg yolk antibody (IgY) production in laying hens in terms of productivity and animal welfare

The antibody production in the egg yolks of immunized laying hens is seen as a way of improving animal welfare compared with conventional production by mammals. Immunoglobulin Y (IgY) technology, however, has still to address welfare issues linked to the widespread use of an adjuvant in vaccines. Currently, Freund's adjuvants, complete (FCA) or incomplete (FIA), remain the standard. This study sought to evaluate various approaches used to enhance egg yolk antibody production in terms of both productivity and avian welfare. The outer membrane protein (OMP) of Salmonella Typhimurium was used as the prototype antigen. At 20 weeks of age, 56 ISA Brown hens, with specific-Salmonella-free status, were divided into seven groups (n = 8) and received an initial intramuscular immunization. Hens in the two negative control groups received phosphate buffered saline (PBS) or FIA alone. Hens in the other groups received 80 μg of Salmonella OMP emulsified with one of the following adjuvants: 200 μl of FIA alone (T1); 200 μl of FIA supplemented with 8 μg of C-phosphate-guanosine oligodeoxynucleotides (CpG-ODN) (T2); and 280 μl of Montanide ISA 70 VG (T4). Birds in the T3 group received the antigen in emulsion with FIA and were given the tested immunostimulatory component (l-carnitine) via their feed (100 mg/kg). A positive control group (PC) received FCA for the first and final immunizations and FIA for the other boosters. Immunization was repeated after 20, 46, 82 and 221 days. Eggs were collected regularly until 242 days after the first immunization and the anti-Salmonella Typhimurium activities in the yolk were determined by ELISA. After 242 days, the birds were euthanized and the injection sites were evaluated for gross and microscopic lesions. Among the tested immunostimulatory approaches, supplementation of FIA with CpG-ODN led to a significant and long-lasting enhancement of the specific antibody response. This treatment was even higher than the positive benchmark using FCA in the first immunization. The study results showed that a clinical examination of injection sites is insufficient for drawing conclusions about the local tolerance of vaccines. Tissue damage was noticeable in all treatment groups. The birds receiving the Montanide adjuvant, however, had fewer and less severe lesions. Given these limited side-effects, Montanide ISA 70 VG could provide the depot effect needed to ensure the immunomodulatory efficiency of CpG-ODN. The association of these two adjuvants could prove a promising alternative to Freund's adjuvants (FA).