Preparation Of Egg Yolk Antibody Against Prv And Study On Its Protective Efficacy

Porcine pseudorabies has been eradicated in some developed countries in Europe and America.In China,the disease can only be prevented by vaccination at present.Since late 2011,a severe PRV outbreak occurred on vaccinated pig farms in most areas of north China,bringing about great economic losses.Unfortunately,there are no effective drugs could cure the disease during the occurrence.Therefore,it is critical to develop effective drugs to control the spread of porcine pseudorabies.The specific egg yolk immunoglobulin(IgY)has attracted considerable attention as a means of controlling infectious disease of bacterial and viral origin.IgY possesses advantages compared with mammalian IgG including low-cost,convenience,high yield,good biosecurity,et al.However,there were no reports about application of IgY against PRV.This subject aims to obtain high titer IgY against PRV through preparation of PRV inactivated vaccine inoculating hens,and then study on its protective effect to provide a scientific basis for IgY in the treatment of porcine pseudorabies in future.The subject including:1 Preparation of specific IgY against PRV and development of IELISA for detecting IgY against PRV24 healthy laying hens of 40-weeks old were divided into 3 groups randomly(group A,B,C)with 8 replications per group.The hens were immunized with prepared inactivated vaccine and the immunization program is as following:group A injected with saline;group B were immunized with inactivated PRV(Bartha-K61)emulsified with oil adjuvant;group C were immunized with inactivated PRV(Bartha-K61)emulsified with Freund’s adjuvant.The second immunization was conducted an interval of 4 weeks after the first immunization.The third immuniza-tion was conducted an interval of 2 weeks after the second immunization.All the groups were totally immunized 3 times with increasing dose(1mL,2mL and 3mL).The eggs were collected and egg yolk was separated from albumen aseptically.Then,IgY was isolated and purified from egg yolk by water dilution,salting-out and ultrafiltration.The purified IgY was detected by SDS-PAGE.Microtiter plates were coated with virions,the purified IgY was used as primary antibody,to establish an IELISA assay.Optimal concentration of the reaction liquid and action time were determined by phalanx titration method.The specificity,sensitivity of antigen-antibody combination and the test reproducibility were evaluated.The variation of IgY titer of the 3 groups was detected by the IELISA assay.The result indicated that approximately 4.6mg IgY was obtained from 10mL egg yolk through isolating,extracting and purifying;The optimal concentration of the coated antigen and the second antibody were 1:100 and 1:4000,respectively,the critical value was 0.170-0.200.The established IELISA assay has a property of good specificity,high sensitivity and good reproducibility;Both group B and group C received high IgY titers.Overall,group C received higher IgY titers compared with group B.2 Neutralization test of anti-PRV IgY in vitro and in vivoThe toxicity test of IgY:IgY was diluted to different concentration in EP tube.With 8 replications per concentration in 96-well cell culture plate,adding PK-15 cells and observing the CPE of cells by fluorescence microscope.The result revealed that IgY with good property of bio-safety did not cause cells to produce CPE.The cell neutralization test of IgY:IgY was diluted to different concentration,and neutralize with 200 TCID50 of PRV in vitro for 1h at 370℃ then adding the PK-15 cells.After observing the CPE of cells,viral DNA was extracted and the copy numbers of the virus were determined by fluorescence quantitative PCR.The result suggested that the PD50 of IgY for PK-15 cells was 0.04,which means that IgY had a good ability of neutralization.When the concentration of the IgY was 575μg/mL,there was significant difference on virus copy numbers between positive control and IgY treatment group.Moreover,virus copy numbers gradually decline with the increase of the concentration of IgY.The neutralization test in vivo of IgY:36 clean-level(Balb/c)mice were divided into 3 groups randomly,with 12 replications per group.The negative control group:The mice were injected with normal saline subcutaneously in the groin,with 0.2mL per mouse.The mice of positive control group were treated with PRV LA strain(TCID50 is 107).While,the test group:the concentrated IgY(25.8mg/mL)was neutralized with PRV LA strain for 1h at 37℃,afterwards,injected the mixture into the mice subcutaneously in the groin with 0.2mL per mouse.Blood samples of the mice were collected(2/12)when the clinical symptoms appeared.Then,DNA from the serum was extracted for PCR to determine whether the mice got viremia.When there was no death of mice appearing,the survival rate was analyzed.Brain,liver,spleen and kidney tissue samples were collected after all the mice were killed and dissected,and then the virus in the 4 tissues were detected by PCR.The neutralization test in vivo of IgY suggested that anti-PRV IgY could offer protection for 80%(8/10)mice and inhibit the proliferation of PRV effectively in mouse tissues.