Establishment Of Indirect ELISA Method And Immunization With Inactivated Vaccine For Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea virus(PEDV)is an infectious enterovirus with high genetic variability.Pigs of different ages can be infected,especially pigs less than 7days old.After infection,severe watery diarrhea,anorexia and weight loss will occur,and the mortality rate can be as high as 100%.In this study,the S2 truncated protein of PEDV was used as the antigen to establish an indirect ELISA detection method for PEDV-specific IgG and IgA antibodies,and a preliminary immune evaluation of the inactivated vaccine prepared by our laboratory was carried out.The specific research content is as follows:1.Establishment of indirect ELISA method for porcine epidemic diarrhea virusThe S2 truncated gene rich in neutralizing epitopes was screened in the early stage and expressed in vitro as an ELISA coating antigen to capture PEDV IgG and IgA antibodies.Goat anti-pig HRP-IgG and HRP-IgA were used as secondary antibodies,respectively.After optimization of the reaction process,two indirect ELISA methods were established to screen PEDV IgG and IgA antibodies in pig serum.The coefficient of variation within and between batches of the two ELISA methods is less than 10%,and the positive serum is tested,and the minimum dilution ratio is 1:800;and for swine fever virus(CSFV)and porcine pseudorabies virus(PRV),Porcine bovine viral diarrhea virus(BVDV)and porcine circovirus(PCV2)positive sera have no cross-reactivity.Comparing the established ELISA method with the commercial PEDV antibody detection kit,the total coincidence rate is higher than90%.This study provides effective technical support for PED pathogen monitoring and inactivated vaccine evaluation.2.Evaluation of the immune effect of inactivated vaccinesThe ELISA method established in this study was compared with the commercial kit,and the inactivated vaccine was evaluated for preliminary immunity.The LT protein is used as an adjuvant and inactivated PEDV is combined to prepare a PEDV inactivated vaccine.Select 2 pregnant sows(no PEDV antibody detected),divide them into immunization group(group A)and control group(group B),and inactivate5m L PEDV by oral administration 2 months and 1 month before delivery.The vaccine and group B each took 5m L DMEM orally.After immunization,the sows showed normal clinical manifestations,and there were no adverse reactions such as detoxification and diarrhea.After immunization,sows in group A can induce specific serum IgG antibodies and mucosal IgA antibodies,both of which show a gradual upward trend.In addition,sows also have higher IgG and IgA antibodies in their milk.Group B sows have always been negative for PEDV antibodies.The piglets of the A and B groups were fed with breast milk for 5 days,and they were divided into 3groups:antibody monitoring group,challenge group and control group.Studies have shown that only 1 piglet in the challenge group of group A developed mild diarrhea,which improved afterwards.The piglets hardly detoxified,and 100%of the piglets were effectively protected;all the piglets of group B had watery diarrhea and the mortality rate was as high as 100%.Pathological anatomy showed that there was no pathological change in the intestinal tissue of the piglets in group A,and there was almost no distribution of PEDV in the intestines;the piglets in group B had thinning intestinal wall,a large amount of light yellow content in the intestines,severe atrophy and shedding of intestinal villi,jejunum and ileum Has a large number of PEDV virus distribution.The final results showed that oral inactivated PEDV vaccine can increase the level of sows'antibodies and enable 100%of piglets to receive passive immune protection.In summary,this study established two indirect ELISA methods to screen PEDV IgG and IgA antibodies in pig serum,and conducted a preliminary immune evaluation of the PEDV inactivated vaccine.The study showed that the piglets obtained by feeding 100% of breast milk Effective passive immune protection.This study provides more effective technical means for further monitoring and prevention and control of the PED pathogen epidemic,and provides theoretical support for the prevention and elimination of PEDV.