Isolation And Identification Of PEDV Epidemic Strain And Preparation Of Quail Yolk Antibody

Porcine epidemic diarrhea(PED)is a highly contagious,intestinal infectious disease caused by Porcine epidemic diarrhea virus(PEDV).The first reported PED outbreak occurred in Europe in the 1970 s and then spread worldwide.At the end of 2010,the disease had a death of over one million piglets in China,with devastating damage to the pig industry.PEDV infections can occur in pigs of all ages,and infections are most serious in piglets,with the morbidity and mortality often reaching 100%.Pig tolerates PEDV infections usually exhibit diarrhea,vomiting and severe dehydration,and the disease course of about 7 days.At present,there is no specific drug to the prevention and control of PED,mainly through vaccination of pregnant sows,and the newborn piglets obtain maternal antibodies to resist PEDV.In addition,commercial vaccines(inactivated or attenuated vaccines)were inoculated to sows by intramuscular injection,which caused the mucosal immune response of pregnant sows to not be effectively stimulated,resulting in insufficient secretion of Ig A antibodies that play a key role in PEDV in breast milk and can not be provided to adequate protection of piglets.Egg yolk antibody(Ig Y)is an immunoglobulin extracted from egg yolk after immunizing poultry with foreign antigen,which can enter the body and specifically bind to the corresponding antigen.Ig Y has stable physical and chemical properties,and has certain resistance to acid,alkali and heat.In recent years,specific Ig Y has been widely used in the prevention and treatment of animal diseases.In this study,the PEDV epidemic strain was isolated and cultured,and the PEDV Freund's adjuvant inactivated vaccine was prepared to immunize egg-laying quails.Then,by comparing different methods of yolk antibody extraction,the best extraction method for Ig Y was screened and optimized.The purified anti-PEDV egg yolk antibody was subjected to in vitro neutralization test and Ig Y physical and chemical properties test.The results obtained were as follows:(1)Isolation and identification of PEDV epidemic strainsAfter processing the small intestine tissues of pigs with positive PEDV nucleic acid,which were inoculated with Vero cells and subcultured continuously to observe the cytopathic changes.The 11 th passage of subculture showed typical syncytial lesions,and the specific bands of PEDV were amplified by RT-PCR,and the sequencing results showed that they were PEDV gene sequences.The indirect immunofluorescence identification results showed that there were viruses in the cells that could interact with anti-N protein monoclonal antibodies.These data shows that a PEDV strain was isolated successfully.(2)Analysis of genetic variation of PEDV isolateThe full-length S1 gene of the PEDV isolate was cloned,and the target fragment was recovered and purified.The results of sequence analysis showed that the isolate was similar to the Sichuan isolate CH-SCNC-2-2015 that was isolated in 2015,Guangdong isolate GD-B,and Anhui isolate AH2012.The S1 gene of isolate had high similarity with 99.1%,and the amino acid similarity is above 98.4%,and the nucleotide similarity of the S1 gene with the standard attenuated strain CV777 and attenuated vaccine strain DR13(South Korea)is 91.9%-92.0%,and the amino acid similarity is 91.9%-92.0%.The similarity ranges from 89.6% to 89.8%.These data shows that the obtained PEDV isolate is an epidemic strain.(3)Preparation of PEDV inactivated vaccine and immunity of quailAfter the venom of the PEDV isolate is filtered and sterilized,which is inactivated by adding 0.1% ?-propiolactone,mixed with the same amount of Freund's complete adjuvant and Freund's incomplete adjuvant respectively,and fully emulsified to make an inactivated vaccine,Perform physical properties,sterility and safety tests.A total of 130 65-day-old egg-laying quails were selected,and each pectoral muscle was injected with 0.2 m L vaccine at multiple points.A total of 3 immunizations were performed,with an interval of 2weeks between each immunization.Freund's complete adjuvant vaccine is the first immunization,and Freund's incomplete adjuvant vaccine is used for the subsequent booster immunizations.After the first immunization,quail eggs were collected every 7 days,and blood was collected to separate serum.The serum and the collected quail eggs were all stored at 4°C.(4)Extraction of yolk antibodies from highly immunity quail eggsThe quail eggs after three immunizations were diluted with water,ammonium sulfate method,and PEG-6000 method to extract the egg yolk antibody.Purity analysis results showed that the yolk antibody extracted by the ammonium sulfate method had higher purity and less impurity protein.It was diluted with water and PEG-6000.The egg yolk antibody extracted by the method has a lot of mixed protein between 30 k Da and 48 k Da.The content of Ig Y protein extracted by water dilution method and PEG-6000 method was9.68 mg/m L and 8.15 mg/m L respectively,and the ammonium sulfate method was 7.56mg/m L,but the highest anti-PEDV antibody titer in Ig Y extracted by ammonium sulfate method was 1.: 5120,so the sulfuric acid method was selected to extract Ig Y.(5)Analysis on the growth and decline of anti-PEDV serum antibody and egg yolk antibody of immune quailThe established ELISA methods was used to detect the anti-PEDV specific antibody in the serum and egg yolk antibody of immunized quail.The results show that the production of specific yolk antibodies lags behind the production of antibodies in the serum.Serum antibodies are tested positive 1 week after the first immunization,while the yolk antibodies are tested positive only 2 weeks after the first immunization.Both antibodies are detected after the third immunization.It reached the highest level in 2 weeks,the high level of serum antibody was maintained for about 10 days,and the high level of yolk antibody was maintained for about 21 days.(6)In vitro neutralization test and physical and chemical properties of anti-PEDV egg yolk antibodyThe neutralization titer of the anti-PEDV egg yolk antibody was 1:111.After storage at 37?56?,p H 3.0?9.0 storage with pepsin for 0.5?2 h,the neutralization ability of Ig Y on PEDV has no significant change.In this study,an epidemic variant strain of PEDV was isolated and a PEDV inactivated vaccine was prepared to immunize egg-laying quails,and finally quail-derived anti-PEDV egg yolk antibodies were obtained.