Specific Chicken Egg Yolk Antibodies against Enterotoxigenic Escherichia coli K99 in Serum and Egg Yolk of Immunized Laying Hens

Enterotoxigenic Escherichia coli K99 is one of the dominant pathogens associated with diarrhea of calves. Immunoglobulin Y (IgY), has been used as an inexpensive alternative to antibiotics for the prevention and therapy of several bacterial infections. The study aimed to prepare IgY antibodies against E. coli K99 and to investigate its in vitro effectiveness. E. coli K99 was grown in the tryptic soy broth, and the bacterial suspension was inactivated by formaldehyde. Thirty White Leghorn hens allocated to control and treatment groups. 1 mL of the prepared bacterial suspension or sterilized physiological serum emulsified with Freund’s complete adjuvant was injected at two weeks interval to the hens in treated or control group. The total IgY was purified with polyethylene glycol (PEG) 6000 from egg yolks (EY). Purified IgY fractions were processed for protein concentrations by Bradford assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze purified IgY fractions. An indirect enzyme-linked immunosorbent assay (ELISA) method was used to measure the specific IgY titers in serum and EY. The ability of non-specific and anti-E. coli K99 IgY antibody at 100 and 200 mg/mL concentration was evaluated by growth inhibition assay in vitro. According to the ELISA data, total IgY concentration in serum and EY of control was relatively constant, while increased in the treatment group (P<0.05). The level of binding activity of specific IgY in serum and EY increased in immunized hens (P<0.05). Specific IgY had the highest activity for bacterial inhibiting growth at the level of 200 mg/mL. These results suggested the prepared IgY antibodies could inhibit E. coli K99 in vitro growth.