Detection of Egg Yolk Immunoglobulin Y ; a Potential Source of Anti-Escherichia coli

1Dietary Supplements and Probiotic Research Center, Alborz University of Medical Sciences, Karaj, IR Iran 2Food Microbiology Research Center, Tehran University of Medical Sciences, Tehran, IR Iran 3Department of Microbiology and Immunology, Alborz University of Medical Sciences, Karaj, IR Iran 4Department of Social Medicine, School of Medicine, Alborz University of Medical Sciences, Karaj, IR Iran


Background
Escherichia coli is a normal resident of the intestines of humans and most animals.Some E. coli strains can cause a wide variety of intestinal and extra-intestinal diseases, such as diarrhea (1)(2)(3).Furthermore, E. coli is one of the major ubiquitous pathogen which causes the food poisoning in notable manner (4).
There have been tremendous efforts to produce numerous new antimicrobial agents to control the bacterial infections; because antibiotic therapy fails in most of bacterial infections because of the increase in antibiotic resistance among bacteria (5,6); therefore, increasing incidence of antibiotic resistance would additionally make problems the treatment of such diseases thus, it is important to look for new therapies to prevent such disease.

Objectives
Recently, scientists focused and intentioned in use of IgY antibodies from eggs of chickens against pathogens for immunotherapy and diagnosis because it has a unique biological activities (7).For prevention of bacterial infections, using IgY has several advantages like it can be easily isolated from egg yolk by the water dilution method on a large scale without using any chemicals or organic solvents.Furthermore, the antibody yield of an egg-laying hen is more than others and more importantly IgY from mammalian blood is time-consuming and expensive (8).Based on the above reports, the present study was formulated with the following objectives; i.To produce immunoglobulin Y (IgY) against E. coli.ii.To identify anti-Escherichia coli immunoglobulin Y in serum by ELISA method.

Experimental Animals
Four 50 week old, white highline chickens were obtained from aviculture and kept in animal house, School of Medicine, Alborz University of Medical Sciences, Karaj.They were used in the study for the production of anti-Escherichia coli antibodies (IgY).

Identification of Escherichia Coli
Escherichia coli were obtained from Microbiology Department, School of Medicine, Alborz University of Medi-cal Sciences, Karaj.The E. coli were isolated form children with acute diarrhea (9).

Preparation of Antigen
Escherichia coli were grown in Brain Heart Infusion Broth overnight at 37˚C.Bacteria was harvested by centrifugation (15 minutes, 3000 rpm), washed three times with PBS (pH 7.2), and re-suspended in PBS at a density of 108 cells/mL by comparing 0.5 McFarland and cells OD at 600 nm was recorded.
For killing the E. coli, formaldehyde was used at concentration of 10% (vol/vol), and suspension was hold at 4°C for 16 hours.In order to remove the formaldehyde, E. coli were washed twice with PBS and re-suspended in sterile PBS.By culturing the E. coli on MHA for 24 hours at 37˚C, complete killing was confirmed and the suspension was stored at 20°C (10).

Development of Anti-Escherichia coli Antibodies in Chickens
Chickens received four injections, one primary injection and three boosters.For the main injection, 500 µL of antigen was emulsified with an equal volume of Freund's complete adjuvant (FCA) using Herbert et al. procedure (11).Then the solution was injected intramuscularly at one site of breast muscle of chickens.Three booster injections of antigen with Freund's incomplete adjuvant (FIA) were given at 14 days interval by the same route of administration.Two mL blood sample was collected from chickens before immunization as preimmune sera.

Serum Separation
Serum was separated by centrifugation for 10 minutes at 3000 rpm and stored at 20°C until use.

Serum's Antibody Titer by Indirect ELISA
The antibodies titer was determined by an enzymelinked immunosorbent assay (ELISA) procedure as described by Sunwoo et al. (12).

Results
The activities of anti-E.coli antibody are detected by ELISA in serum from laying hens as shown in Figures 1, 2 and 3.
The titer of the specific antibody produced in immunized hens increased rapidly from the first injection and reached a peak after second booster injection at the fourth week and was maintained stable for two weeks.However it decreased after last booster injection that this subject shows in order to achieve the stability in the titer of antibody, require repeated booster injections (Figure 1).Furthermore comparing serum dilutions with control group (preimmune sera) indicate that up to 1.10000 dilution antibodies present in serum, which shows high amount of antibodies are produced in immunized chickens (Figures 2 and 3).

Discussion
Administration of IgY to host pathogens is an attractive approach to establish protective immunity, especially against gastrointestinal pathogens in human; furthermore, scientists reported that, IgY has been used for suppression of growth of food-borne pathogens (13).In addition, the specificity of avian antibodies often differs significantly from that of comparable mammalian antibodies.An important consequence of this is that the Rheumatoid Factor (RF) cannot bind with IgY.RF is a major source of interference in many immunoassays, being an autoantibody that reacts with the Fc portion of mammalian IgG.The disease usually associated with RF is rheumatoid arthritis, but RF is also present in serum from patients with many other diseases.Most immunoassays use mammalian polyclonal or monoclonal antibodies, which are prone to RF binding, thus giving false positive results.However, RF is not able to bind to the IgY molecules; therefore, chicken antibodies can be useful in assays (e.g.nephelometry, latex-aggregation, or ELISA) were RF could interfere.Apart from the biological differences (RF recognition) the difference in molecular structure gives rise to a variety of physicochemical differences (solubility, affinity for adsorption, isoelectric point, etc.).The heavy chains (H) of IgY are larger than, and antigenically distinct from, those of mammalian IgG (14).
In the present study, the activity and titer of specific immunoglobulin Y in serum, which were determined by ELISA, showed the presence of antigen specific antibodies for the specific pathogenic bacteria.As others also reported, ELISA is a sensitive technique to find out the antibody titers during different purification processes (15).Further studies have to be conducted to evaluate the potency of these antibodies in treatment of variety of diseases.The outcome of this research work would be an alternative to the current antibiotic treatments.As mentioned earlier, IgY These IgY treatments has a variety of significant like it is safer, more efficient and less expensive as compared to conventional mammalian antibiotics for controlling pathogenic bacteria like E. coli.Recently, successful progresses in industrialization of IgY has been achieved in Japan, where IgY as a bioactive ingredient in food, nutraceuticals, cosmetics and other sectors is applied (16).Further studies have to be conducted to evaluate the potency of these antibodies in treatment of variety of diseases.The outcome of this research work would be an alternative to the current antibiotic treatments.