Preparation Of Yolk Antibody Against Piglet Colibacillosis And Its Preliminary Application

Piglet colibacillosis is one of the common diarrhea in pig-raising industry in the whole world,which is caused by enterotoxigenic Escherichia coli.Strains of K88,K99,987 P and F41 were responsible for it,,mainly adhere to the small intestinal epithelial cells,colonize and reproduce in large numbers,thus playing a pathogenic role.Due to the complex serotypes of E.coli,the extensive usage of antibiotics leads to the mutual transmission and spread of drug-resistant plasmids,and their drug resistance is significantly enhanced,which brings great difficulties to prevention and treatment.The accumulated data indicate applicaiont of egg yolk antibody(Ig Y)can replace for antibiotics to maintain the health and growth performance of piglets,which brings hope for the prevention and treatment of animal colibacillosis.Based on the above situation,in the study four inactivated E.coli strains K88,K99,987 P and F41 were administed into laying hens multiple for four times;An indirect ELISA detection method was establishment to monitor the yolk antibody levels at various stages of the immune cycle,the stability of the yolk antibody also was evaluate;Fnally,the yolk antibody is initially applied to piglet colibacillosis to evaluate its protective effect.The specific research contents are as follows:Methods:(1)Immunization of laying hens: mix the four E.coli strains with the immunopotentiator at a 1:1 ratio to prepare an immune antigen,immunize the laying hens at a dose of 1 m L/head,and immunize four times.Immune once every 10 days.(2)Extraction and purification of egg yolk antibody: collect immunized eggs,extract and purify by water dilution method combined with saturated ammonium sulfate two-step separation method,and identify egg yolk antibody by protein concentration determination kit and SDS-PAGE electrophoresis method.(3)Establishment of indirect ELISA detection method: Four types of E.coli strains were ultrasonically broken into bacterial liquid protein,and the supernatant was taken as detection antigen.A series of optimal reaction conditions were screened by matrix method.(4)Stability determination of yolk antibody:the established indirect ELISA detection method was used to determine the stability of lyophilized powder under different temperatures and acid-base conditions.(5)Initial application of yolk antibodies: 15 weaned piglets that were not immunized with E.coli vaccine were tested for prevention and treatment,and a control group was set up to protect the piglets from diarrhea,blood routine indicators and weight gain Comprehensive analysis of the effect.Results:(1)The yolk antibody was successfully extracted and purified by water dilution method and saturated ammonium sulfate two-step separation method,and the yolk antibody was identified by SDS-PAGE electrophoresis,and the purity can reach about 96%;determined by protein concentration kit The purified yolk antibody concentration was 1.05mg/m L.(2)The yolk antibody was measured by indirect ELISA detection method.The antibody level began to rise slowly at 10 days after the first immunization.At 60 days,the antibody level reached a peak.Its antibody titer was 1:25 600,and the antibody level appeared for a short time at 70 days.It decreased slightly and then recovered to the highest level;the stability test results showed that the yolk antibody had good stability under the conditions of 65 ℃ for 15 min,37 ℃ for 24 h and p H 3～10.(3)Immune protection test:Comprehensive analysis is carried out through the status of piglet feces,blood routine indicators and weight gain.The results show that the specific yolk antibody has a good preventive effect on piglet E.coli disease,and its protection rate is 100%;the treatment group piglets are artificially infected with E.coli,and the prevalence rate is 100%.Treated with good results,the cure rate is 100%.