Production And Features Of Egg Yolk IgY Against Hafnia Alvei

Bee septicemia is a common bee disease caused by Hafnia alvei.Generally,the death rate of infection is as high as 90%,and it is considered to be one of the most serious diseases that endanger bee breeding.At present,most of the antibiotics such as cephalosporins and tetracyclines are used to treat bee diseases caused by the bacteria,which will cause the bees to develop drug resistance and residual antibiotics in the honey to threaten human health.Immunoglobulin of yolk(IgY)originated from oviparous animals.As a serum antibody,it is mainly synthesized in the blood and then transferred to the egg yolk.It has the characteristics of short development cycle,low cost,and stable physical and chemical properties.This research is based on Hafnia alvei and explored the optimal conditions for cultivating Hafnia alvei.IgY was prepared by immunizing laying hens with inactivated Hafnia alvei as the antigen.After IgY was separated and purified,it passed the test The antibody titer,purity and the content of specific antibodies in it are used to evaluate IgY;the indirect ELISA detection method of Hafnia alvei has been established and optimized;by investigating the results of in vitro antibacterial,the study of specific IgY on Hafnia alvei in vitro The growth inhibitory effect of niobium;the effect of specific IgY on the treatment and prevention of honeybees infected with Hafnia alvei was investigated through oral administration.The main results are as follows:(1)The optimal composition of the Hafnia alvei culture medium is as follows:carbon source is 2%glucose,nitrogen source is 2%peptone,sodium chloride is added4%,and the order of primary and secondary factors is peptone>sodium chloride>glucose.The results of single factor and orthogonal analysis of growth conditions showed that the optimal conditions for the cultivation of Hafnia alvei were a temperature of 35°C,a p H of 8,and a shaker speed of 200 rpm;(2)Five methods were used to separate and purify the specific IgY.Among them,the specific IgY separated and purified by the polyethylene glycol precipitation method had high purity,and the concentration of IgY was 9.94 mg/m L.The ELISA results at the7th week after the first immunization showed that the titer of the stock solution of egg yolk antibody was 1:3276800,while the titer of the lyophilized powder was 1:819200;(3)Establish and optimize the indirect ELISA detection method for Hafnia alvei specific IgY.The established indirect ELISA assay method is improved by using the known specific IgY and non-specific IgY against Hafnia alvei.The improved ELISA procedure is as follows:Coat with the inactivated Hafnia alvei diluted with the coating solution,dilute to 1×10~6 CFU/m L,place at 37°C for 1 hour and then overnight at 4°C.Add 0.2%BSA for blocking,and block for 2 h at 37°C.The primary antibody is specific IgY reconstituted in 1:1000 lyophilized powder,reacted at 37°C for 1 h.The enzyme-labeled secondary antibody was diluted 1:8000 and reacted at 37°C for 1 h.The substrate develops for 10 min.According to the optimized ELISA procedure,the fragmented supernatant of Hafnia alvei with different dilution gradients was tested,and the positive cut-off value of this method was determined to be 0.5989,and the negative cut-off value was 0.6471,which was between 0.6 and 0.65.Suspicious results,suspicious samples should be repeated.If the re-examination result is still suspicious,it is judged as negative.The coincidence rate results are all greater than 95%.When the dilution of the strong positive sample is 1:3200,the result is still positive.When the dilution of the weakly positive sample is 1:800,the result is still positive;(4)The in vitro antibacterial results of specific IgY on Hafnia alvei showed that compared with the control group and non-specific IgY,10 mg/m L specific IgY could significantly inhibit the growth of Hafnia alvei in liquid medium.Animal experiments show that,compared with the control group and non-specific IgY,the bee bread containing specific IgY has obvious protective and therapeutic effects on the bees after challenge,and the specific IgY has obvious protection on the bees infected with Hafnia alvei Its protection rate reached more than 95%,and at the same time through the infection of Bacillus larvae and Salmonella(the survival rate was less than 20%),it also verified the specificity of its protection effect.