Ex Vivo Evaluation of Egg Yolk IgY Degradation in Chicken Gastrointestinal Tract

Egg yolk antibody (immunoglobulin Y, IgY), due to its unique features (e.g., cost-effectiveness for mass production), is emerging as a promising passive immune agent and alternative to antibiotics to combat infectious diseases, particularly in livestock. Oral administration of egg yolk IgY is the most common and convenient route that has been extensively investigated for controlling enteric pathogens. However, the in vivo stability of egg yolk IgY in the gastrointestinal (GI) tract, a critical issue for the success of this approach, still has not been clearly elucidated. Our recent study showed instability of orally administered egg yolk IgY in chicken GI tract, as demonstrated by both in vivo and ex vivo evidence. To better understand the magnitude and dynamics of instability of egg yolk IgY in vivo, in this study, we conducted comprehensive ex vivo analyses by spiking hyperimmune egg yolk IgY in fresh GI contents collected from five broilers at each sampling age (2, 4, or 6 weeks). The pH in gizzard slightly increased with age from 2.4 to 3.0, while the pH in the small intestine was around 5.8. ELISA analysis indicated that a short time of treatment (30 or 60 min) of IgY with the gizzard contents from the chickens at 2, 4, and 6 weeks of age greatly reduced specific IgY titer by over 8, 6, and 5 log2 units, respectively, when compared with saline control. However, small intestine content only had a mild effect on egg yolk IgY, leading to 1 log2 unit of reduction in IgY titer upon 30 min of treatment. Consistent with these findings, SDS-PAGE and immunoblotting analyses provided direct evidence demonstrating that egg yolk IgY could be drastically degraded to undetectable level in gizzard content upon as short as 5 min of treatment; however, the IgY was only slightly degraded in small intestine content. Immunoblotting also showed that treatment of IgY with HCl (pH 3.0) for 60 min did not affect its integrity at all, further supporting the enzymatic degradation of IgY in gizzard. Collectively, egg yolk IgY could be substantially degraded in chicken gizzard, highly warranting the development of effective approaches, such as encapsulation, for the controlled release and protection of orally administered egg yolk IgY in livestock.

Chicken Egg Yolk Antibody (IgY) Protects Mice Against Enterotoxigenic Escherichia coli Infection Through Improving Intestinal Health and Immune Response

Chicken egg yolk antibody (IgY), considered as a potential substitute for antibiotics, has been used for preventing pathogens infection in food, human and animals. This study investigated effects of IgY on growth, adhesion inhibitory and morphology of enterotoxigenic Escherichia coli (ETEC) K88 in vitro, and evaluated the protective effects of IgY on intestinal health and immune response of mice infected with ETEC in vivo. Sixty pathogen-free C57BL/6J (4-6 weeks of age) mice were divided into six treatments: control (neither IgY nor ETEC infection), ETEC infection, ETEC-infected mice treated with 250 μL of high-dose (32 mg/mL), medium-dose (16 mg/mL) or low-dose (8 mg/mL) anti-ETEC IgY, or ETEC-infected mice treated with 250 μL of non-specific IgY (16 mg/mL). Anti-ETEC IgY inhibited ETEC growth, reduced adherence of ETEC to intestinal epithelial cells J2 and damaged the morphology and integrity of ETEC cell. Oral administration of anti-ETEC IgY effectively ameliorated ETEC-induced clinical signs, reduced ETEC colonization and intestinal permeability, alleviated inflammatory response through reducing the production and expression of proinflammatory cytokines, improved intestinal morphology, and inhibited excessive activation of the mucosal immune response of challenged mice. The overall protective effects of high-dose and medium-dose anti-ETEC IgY against ETEC infection were more effective. These results suggest that anti-ETEC IgY may function as a promising novel prophylactic agent against enteric pathogens infection.

PSVIII-2 Antibody yield for polyclonal egg yolk antibody (IgY) production in laying hens

Antibody production in egg yolks of immunized laying hens is an alternative to conventional mammalian production. Antibody yield peak and duration have not been described for immunoglobulin Y technology using Freund’s incomplete adjuvant (FIA) and C-phosphate-guanosine oligodeoxynucleotides (CpG-ODN) without the inclusion of Freund’s complete adjuvant for enhancing the immune response to an interleukin-10 (IL-10) peptide. This study sought to describe the antibody titer production for an 8 amino acid sequence from the surface of the bovine IL-10 protein (VMPQAENG) as the antigen emulsified with CpG-ODN and FIA in phosphate buffered saline (PBS). 60 hens were assigned to receive the complete vaccine (Peptide), 20 received the vaccine without the IL-10 peptide (Control), and 8 received a PBS injection (Blank). Hens were immunized with 0.25 mL in 4 locations, each breast and each thigh on days 1, 15 and 29. The complete vaccine delivered 0.6 mg IL-10 peptide, 8 µg CpG-ODN, and 0.33 mL FIA per hen on each vaccination day. Eggs were collected regularly until 175 days after the first immunization and the anti IL-10 peptide activities of the yolk were determined by ELISA. Egg titers by treatment were analyzed with a repeated measures ANOVA in SAS. The supplementation of FIA with CpG-ODN produced high titers, of over 100 µg of antibody per mL of yolk (µg Ab/mL yolk), around day 33 through day 76, with a slow decline through day 175 when average titers remained above 40 µg Ab/mL yolk. Peptide egg titers were significantly higher than Blank or Control titers from day 31 though day 175 (P < 0.0001). Titers recovered from Marcq et al. (2015) with similar methods were 1.5 to 7 times lower than these results over the same number of days.