Immunization Test Of Genetically Engineered Inactivated Vaccine Of Porcine Epidemic Diarrhea Virus

Porcine Epidemic Diarrhea Virus(PEDV)is one of the main pathogens that cause diarrhea in pigs worldwide.Especially in the past ten years,PEDV has caused huge economic losses in the pig industry.PEDV has a high infection rate for pigs of all ages,causing symptoms such as diarrhea and dehydration,which are much more severe with piglets under 7days of age with high morbidity and mortality.At present,the most efficient way to prevent and control PEDV is vaccine immunization,which includes active immunization and passive immunization.The measures to protect adult pigs from PEDV infection are mainly through active immunization,but the way to prevent and control PEDV in young piglets is mainly through passive immunization.In this study,an immunization efficacy study was carried out to evaluate an inactive PEDV vaccine candidate developed in our lab.The study is composed of two parts: one with 30-day-old piglets,types of adjuvant and dosage of vaccination was investigated.Then,with pregnant sows immunization test was carried out and passive immunization effect was evaluated with their newborn piglets.The first experiment in the study was to establish an indirect ELISA to detect PEDV antibody induced by the vaccine candidate.To this aim,a segment of PEDV S1 protein(139aa)expressed by prokaryotic cells was used as the coating antigen and sheep anti-pig IgG-HRP was used as the secondary antibody.Reaction conditions were optimized and specificity,sensitivity and repeatability tests were carried out.The specificity test showed that the established method had no cross-reaction with the positive serum of TGEV,PoRV and PDCoV,which are common viral pathogens causing porcine diarrhea.The sensitivity test showed that the method could detect 800 times diluted IgG positive serum.The coefficient of variation in repetitive experiments was less than 10%.The second experiment was to select a suitable adjuvant for the vaccine candidate.To this aim,21 30-day-old piglets were randomly divided into 6 groups: DMEM group(3),aluminum hydroxide adjuvant group(3),201 adjuvant group(4),15 A adjuvant group(4),Gel02 adjuvant group(4),and 715 adjuvant group(3).The vaccine reagent was applied intramuscularly and twice,two weeks apart.After vaccination,IgG antibody levels of pigs in each group were monitored,and it was found that the antibody levels of pigs in the 15 A adjuvant group were significantly higher than those in the other adjuvant groups.The antibody levels of alumina hydroxide adjuvant group and 206 adjuvant group substantiallyelevated,but not to a level positive.There was no significant variation in antibody levels in other groups,so 15 A adjuvant was selected for subsequent studies.The third experiment was to explore the dose-effect relationship with the inactivated vaccine candidate.To this aim,17 30-day-old piglets were divided randomly into 6 groups:DMEM group(2),0.6mL group(3),1mL group(3),2mL group(3),4mL group(3)and 8mL group(3).The animals were vaccinated intramuscularly and twice,at an interval of two weeks.ELISA of IgG showed that the value of 8 mL group was obviously higher than that of other groups,while that of 4 mL and 2 mL group was slightly lower.Most of the animals in the groups of 0.6 mL and 1 mL did not turn to positive in the process.Therefore there is a dose-effect relationship with the tested dosages of the vaccine candidate.The fourth experiment was to evaluate the passive immunological protection of the inactivated vaccine candidate to newborn piglets.To this aim,8 pregnant sows were divided randomly into two groups: the immunized group(4)and control group(4).After being born,piglets were allowed to suck colostrum for 4 days.8 randomly selected piglets from vaccinated and control sows were respectively employed for challenging(designated as immune challenge group and non-immune challenge group.13 piglets which were kept staying with their vaccinated or non-vaccinated mothers were used for monitoring the antibody level during the whole process without challenging.Piglets from both the immune challenge group and the non-immune challenge group were challenged simultaneously with a wild intestinal strain pedv-xs12.Clinical observation showed that piglets in the immune challenge group developed diarrhea after the challenge,but could keep alive within the initial5 experimental days,necropsy showed lesions in some intestinal segments.Piglets in the non-immune challenge group however developed more severe diarrhea after the challenge,followed by rapid dehydration,loss of appetite,and lethargy,eventually all died within 2 days of symptom onset.Autopsy of piglets dead from diarrhea showed that the intestine took on pathological changes of typical PED: small intestine aerated and thinned,with severe lesions on tissues as well.Piglet antibody testing results showed there was an obvious correlation between the IgG level and the after challenging state of the animals.Although all the piglets had diarrhea in the early days after the challenge,which might be due to the high challenging dosage or short ingestion time of sow's milk,the physiological state of the piglets in the immune challenge group was much better than the non-immune challenge group.This indicates that the PEDV inactivated vaccine could induce active immune effect by intramuscular injection in sows,and could also confer obvious passive immune protection to the piglets after ingestion of colostrum.