Production and Purification Immunoglobulin against E. coli in Egg Yolk

Chicken is the only avian species in which polyclonal antibodies, like IgG is transported from the hen to the egg yolk in a similar manner as the transport of mammalian IgG from the mother to the fetus. Immunoglobulin Y in the chicken is transported to the egg and accumulates in the egg yolk in large quantities. IgY is an egg yolk antibody that has been used widely for treatment and prevention of infections in humans and animal. IgY is used for passive protection of the pathogen infections such as Escherichia coli, bovine and human rotavirus, bovine coronavirus, salmonella, staphylococcus and Pseudomonas. IgY is a promising candidate as an alternative to antibiotics. Eschericha coli strains of serotype O157: H7 belongs to a family of pathogenic E. coli called enterohemorrhagic E. coli (EHEC) strains responsible for hemorrhagic colitis, bloody or non-bloody diarrhea, and hemolytic uremic syndrome in humans. This strain of E. coli pathogenises by adhering to host intestinal epithelium and forming bacterial colonies. The purpose of this study was to produce and purify immunoglobulin Y against E. coli O157:H7 and develop specific polyclonal anti E. coli antibody in the egg yolk.
Sixteen-week-old laying hens (Mashhad, Iran) were kept in individual cages with food and water ad libitum. Immunization of hens was performed by intramuscularly injecting killed E. coli O157: H7 with an equal volume of Freund’s complete adjuvant into two sides of chest area (Sigma, USA) for the first immunization. Two booster immunizations followed up using complete and incomplete Freund’s adjuvants in two weeks interval. Freund’s adjuvant without antigen was injected to the control group. Two weeks after the last injection, the eggs were collected daily for eight weeks, marked and stored at 4 ºC. In order to IgY purification, eggs were collected. Purification of IgY from egg yolk was based on Polson and using PEG6000. Finally, the presence of antibody IgY was confirmed using SDS-PAGE. Purification of IgY was carried out by polyethylene glycol precipitation method using PEG 6000 powder (Merck, Germany) based on method of Polson. The purified IgY against E. coli was separated using 10% SDS-PAGE. In order to investigate the effect of the specific anti-E. Coli antibody, mice (Razi, Institute of Iran) were randomly distributed into five experimental groups (6mice/group). The mice were kept in conventional animal facilities and received water and food ad libitum. All animal care and procedures were in accordance with institutional policies for animal health and well-being. Experimental groups were including group 1 (mice received IgY orally in drinking water 72 hours before intraperitoneal injection of bacteria and then injected intraperitoneally with 0.5 ml of bacteria E. coli O157: H7), group 2 (mice received IgY orally in drinking water 72 hours before the injection and then injected intraperitoneally with 0.5 ml of deionized water), group 3 (0.5 ml of E. coli O157: H7 incubated with 0.5 ml of the specific anti-E. coli IgY and then 0.5 ml of the incubated solution injected to mice intraperitoneally), group 4 (mice injected with 0.5 ml of IgY) and group 5 (mice received 0.5 ml of E. coli O157: H7).
We obtained specific egg yolk antibody against E. coli O157: H7 by immunizing hens with the killed E. coli O157: H7 antigen. The results showed that the IgY was successfully purified from egg yolk. SDS-PAGE analysis showed presence of protein bands 27kDa and 67 kDa of IgY, which correspond to IgY light and heavy chains. Effects of IgY on mice showed that mice received IgY orally in drinking water 72 hours before intraperitoneal injection were protected against bacteria. Also, when specific anti-E. coli IgY was incubated with E.coli O157: H7 for 24 hours and then it was injected to mice led to mice protected against bacteria. The results of our study were agreement with the results of Chae et al (2007). We indicated mice immunized with specific anti-E. coli IgY could be protected against E. coli O157: H7. This phenomenon could be due to specific binding activity of IgY with bacteria that led to the inhibition of bacterial growth E. coli O157: H7.
The effectiveness of IgY in suppressing the activity of E. coli O157:H7 was indicated in our study. This could be inferred from the results of the current study that IgY in the egg yolk could prevent greater economic losses due to human and animal health from pathogenic bacteria such as E. coli O157:H7. These finding indicated that egg from immunized hens are potentially useful source of passive immunity.