Studies On The Preparation Of Immunoglobulin Y From Egg Yolk Against Piglet Diarrhea

Enterotoxigenic Escherichia coli (ETEC) is one of the pathogenic bacteria that can cause piglet diarrhea. Several serogroups such as K88, K99, 987P and F41 have been isolated and identified from other ETEC cultures. A study described in this thesis dealt with the combination of K88-K99-987P and K88-K99-F41 with different commercial adjuvant [Freund's adjuvant, White oil adjuvant, Al (OH) 3 adjuvant, and PBS] that were immunized onto the laying chickens, respectively. The preparation methods of the antibody, IgY from egg yolk, were investigated against piglet diarrhea. The purification methods of IgY and other systemic researches on the bioactivity and characteristics of IgY were considered in this thesis.The culture medium used for enumeration of K88, K99, 987P and F41 to produce fimbria was Minca. For the determination of growth curves of all these strains, it was concluded that the best incubation periods were all 12 h. After continuous immunization, both the Freund's adjuvant vaccines and the White oil adjuvant vaccines were found to induce the chickens to produce stable and high level of IgY compared to the Al (OH) 3 adjuvant vaccines and PBS vaccines, respectively.A procedure was therefore developed for the extraction of IgY by using water dilution combined with floc clarification reagent. The recovery of water-soluble protein was 84.47%. The result of SDS-PAGE showed that molecular weights of IgY, light chain and heavy chain were 188000, 28400 and 66500, respectively. By using water dilution, floc clarification and affinity chromatography method, the recovery of IgY was 17.92%. Likewise, the IgY recovery of 16.43% was obtained for the method of water dilution, floc clarification, (NH4)2SO4 precipitation and affinity chromatography. The IgY content per egg was 1.22‰(w/w).The IgY-K88-K99-987P and IgY-K88-K99-F41 titers for Freund's adjuvant vaccines were 32 times higher, whereas for the other two White oil adjuvant vaccines were 16 times higher than the titer obtained from the non-immunized chickens.There was no significant difference found in the activity of either purified IgY or unpurified IgY when the temperature ranged from 20–50°C. However, when the temperature increased further, the resistance of unpurified IgY against heat was better. The testing of IgY against acidity (range: 2.0– 4.0) and/ alkalinity (8.0– 10.0) resulted in low activity when the temperature was high. The purified IgY was more sensitive to the changes in pH at 60°C than at 20 and 37°C. The changes in pH had a distinct effect on the activity of IgY at 37°C when the pH ranged from 2.0 to 4.0 and from 8.0 to 10.0, respectively. The test of IgY hydrolyzed by pepsin showed that the resistance of IgY to pepsin hydrolyzation was related to pH. The IgY hydrolyzed by pepsin was significant at pH 2.0 and 3.0; whereas was not significant...