| Porcine epidemic diarrhea virus(PEDV)is one of the major pathogens causing diarrhea in swine worldwide.Especially in the past decade,G2 PEDV caused huge economic and piglet losses in the pig industry.PEDV has a high infection rate in all ages,which can cause symptoms such as diarrhea and dehydration.Vaccine immunization is currently the most important way to prevent PEDV including active immunity and passive immunity.PEDV has the most serious impact on newborn piglets causing high morbidity and mortality.Passive immunity is important for newborn piglets.Therefore,this study used pregnant sows and their piglets to study the passive immunity of the inactivated vaccine against G2 PEDV.Inactivated vaccines usually cause specific antibodies to the body.To elucidate the relationship between IgA,IgG antibody and immune protection in piglets and sows during passive immunization,this study first established an indirect ELISA assay for testing PEDV-specific IgA and IgG antibodies in serum.After expressed and purified,the PEDV S1 protein was used as the coating antigen.And the HRP-conjugated goat anti-pig IgA was used as the secondary antibody.With reaction conditions were optimized,the iELISA was established for the detection of anti-PEDV G2 genotype IgA antibody,followed by the specificity,sensitivity and repetitive tests.The specificity test showed no cross-reaction with those positive sera of porcine TGEV,PoRV,PDCoV and PCV2.The sensitivity test showed that the method can detect 400-fold diluted IgA positive serum and 800-fold diluted IgG positive serum respectively.The coefficients of variation(CV)of the intra-and inter-batch repetitive tests were both less than 15%.The iELISA and indirect immunofluorescence method(IFA)were used to simultaneously detect clinical samples,and the results showed that the total coincidence rates of IgA,IgG antibody indirect ELISA and IFA were 98.6% and 96.5%,respectively.To elucidate the passive immunity of G2 PEDV,the pregnant sows were divided into two groups(A and B)and immunized with DMEM and PEDV inactivated vaccine on days 40,20 pre-farrowing,respectively.The newborn piglets were fed colostrum,and the piglets were randomly divided into three groups: the challenge,the control,and the monitoring group.Part of piglets was infected by G2 PEDV LNct2 a strain F13 after 5 days of breastfeeding.Group A sows were only injected with DMEM as a control group,and piglets were also infected with PEDV after 5 days of breastfeeding.Clinical symptoms showed that piglets born in non-immunized sows had signs of depression and diarrhea after 24 hours of infection,and the morbidity was 100% within 5 days.The results of the abdominal examination showed that the intestinal tissue became thinned and filled with yellow liquid and food residue.RT-qPCR results showed that piglets of this group excrete more virus and the viral RNA was detected in the intestinal tissues after infection.The infection of intestinal epithelial cells by PEDV was also observed by immunohistochemistry(IHC).In contrast,piglets born in immunized sows received up to 89.5% protection,while only 10.5% of piglets showed normal intestinal tissue and mild diarrhea.There was almost no excretion of PEDV virus in piglets born in group B sows,and the viral RNA content in intestinal tissues was extremely low.The ELISA results showed that the levels of IgG and IgA antibodies in pig serum were strongly correlated with the level of neutralizing antibodies(NA),indicating that the houhai acupoint immunity by PEDV inactivated vaccine can induce higher levels of antibody response and form immune protection for piglets.In summary,this study preliminarily demonstrated that the PEDV inactivated vaccine stimulated sows to produce high levels of IgG,IgA and neutralizing antibodies,and provided passive protection for 5-year old piglets,which provided a theoretical basis for further research on the mechanism of immune protection. |
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