Preparation And Preliminary Application Of Anti-PoRV And PEDV Dual Yolk Antibody

Diarrhea of piglet is the most harmful digestive system disease to pig industry in recent years,causing a large number of piglets death or production performance decline,seriously affecting the production performance and economic benefits of pig farms.Porcine epidemic diarrhea（PED）and porcine rotavirus（PoRV）infection caused serious death of newborn piglets,but the lack of effective prevention and control and treatment means,once the disease will be difficult to control,infected pigs often in a short time after the occurrence of diarrhea a large number of deaths,serious mortality even up to 100%.The most commonly used strategy for the prevention and control of PED and PoRV infection is prenatal vaccination of pregnant sows.Newborn piglets can obtain specific protective antibodies from the colostrum,but the practical effect is not satisfactory.The reason is that once newborn piglets are infected with porcine Epidemic diarrhea virus（PEDV）or porcine rotavirus（PoRV）,It will soon cause damage to the villi of the small intestine,and there will be severe diarrhea and dehydration.The maternal antibody cannot prevent the infection of the virus,so the protection degree is limited.Vaccination of newborn piglets is also a strategy,but the piglets’immune system is not sound,and it takes a long time to produce a protective immune response.However,the disease course caused by the above viruses is short,and the infected piglets will become sick and die before the immune response is produced.In addition,there are no specific drugs for the treatment of viral diseases;Moreover,the emergence of PEDV and PoRV mutant strains in recent years has also greatly affected the immunization effect of the vaccine,and the immune antibodies produced by the vaccine strain cannot completely resist the damage of the epidemic strain（wild strain）.Egg yolk antibody（Ig Y）is produced by immunizing specific pathogens of laying hens.It is stable and resistant to acid and base.A number of studies at home and abroad have confirmed that the application of specific yolk antibody to infected pigs can greatly reduce the incidence and severity of infected pigs.Specific egg yolk antibody has been paid more and more attention in the prevention and control of animal disease due to its advantages such as clear effect,simple preparation and good safety.（1）Isolation and identification of porcine rotavirus epidemic strainsBased on the detection and analysis of the main viruses causing diarrhea of piglets in pig farms,this study intends to isolate and identify the detected piglet diarrhea viruses and analyze their genetic variation.The specific egg yolk antibody was prepared for infection protection test.It provides basic data for the epidemiological study of viral diarrhea of piglets and a new preparation for the prevention and control of diarrhea of piglets.This study obtained the following results:（1）Detection,isolation and identification of porcine diarrhea virusMajor diarrhea viruses were detected in 25 pig farms in 4 cities of Shaanxi Province,and the positive rate of PEDV was the highest,with a positive rate of 18.5%（22/119）,followed by that of PoRV 16.0%（19/119）,and the positive rate of PEDV mixed infection with PoRV was 10.9%（13/119）.Neither TGEV nor PDCo V was detected.After treatment,19 fecal samples tested positive for PoRV and 22 feces tested positive for PEDV were inoculated into MA-104 and Vero cells for continuous culture and observation of cytopathosis.When cultured to the 7th generation,PoRV positive disease cells showed pathological changes,PCR detection and IFA identification showed that the isolated strain was PoRV,and the TCID₅₀of this strain was 10^-6.11/0.1 m L.PEDV is not isolated.（2）Genetic evolution analysis of PoRV isolatesWhole genome sequencing and analysis showed that the VP6 gene of PoRV was I5,which had the highest nucleotide homology with porcine rotavirus A NMTL（JF781162.1）（98.03%）.The genotype of VP7 gene was G9,which had the highest homology with TM-a（KC113248.1）（94.69%）.The genotype of VP4 gene was P[23],and the isolated strain was widely circulated in humans and pigs in recent years.（3）Preparation of yolk antibody and analysis of its levelPoRV and PEDV venom were filtered for sterilization and concentrated,thenβ-propanolactone was added（the final concentration was 0.1%）for inactivation.PoRV+PEDV vaccine was prepared from the mixture of PoRV and PEDV virus,and then used for immunization of laying hens.Twenty-eight laying hens in peak laying period were inoculated with two inactivated vaccines.Eggs with high titer after triple immunity were collected to extract yolk antibody.The results showed that the extracted yolk antibody concentration was 13.26 mg/m L.The neutralizing titer of the extracted anti-PoRV yolk antibody and anti-PEDV yolk antibody was 1:273 and 1:307 respectively by fixing virus diluted yolk antibody.In the acidic environment with p H 3,the OD₄₅₀value of purified yolk antibody can still be maintained at about 0.9.In the alkaline environment with p H 10,the OD₄₅₀value of Ig Y is about 1.1.The test results show that the yolk antibody is relatively stable in the weak acid or weak base environment;The ELISA method was established to determine the antibody titers in serum and yolk of immunized laying hens.The level of PoRV antibody in serum increased earlier than the level of PoRV antibody in yolk.The level of PoRV antibody in serum peaked on the 42nd day after immunization and began to decline after maintaining 30 days.Compared with PoRV yolk antibody,the production of antibody in serum was earlier,but the level of antibody in yolk was higher than that in serum after immunization 63 days.PEDV serum antibody and yolk antibody were observed.Similarly,the initial serum antibody was higher than the level of yolk antibody,and the PEDV yolk antibody was higher than the serum antibody level after 56 days of immunization.（4）Challenge protection test of yolk antibody in pigletsThe experiment was divided into 3 groups.The challenge group was given PoRV and PEDV virus mixture at 5 days of age.At 3 days of age,the treatment group was given freeze-dried powder with double yolk antibody daily for 5 days,and at 5 days of age,PoRV and PEDV virus mixture was orally administered.The negative group was fed normally.All piglets were not fed colostrum.Survival rate,enterovirus content and histopathological section results were used to evaluate the effect of yolk antibody.The results showed that the survival rate of the yolk antibody treatment group was 66.7%and that of the challenge group was 0%.The virus content of each small intestine in the treatment group was significantly lower than that in the challenge group.The small intestinal histopathologic sections of the yolk antibody treatment group showed a small number of villi breakage,the shape of which was basically normal,while the challenge group showed pathological changes such as small intestinal mucosa shedding and villi breakage.The results showed that the yolk antibody could protect piglets against virus to a great extent.In conclusion,the detection rates of PEDV and PoRV were the highest among the 25pig farms,which were 18.5%and 16.0%,respectively,and the mixed infection rate of PEDV and PoRV was 10.9%.One PoRV strain was isolated from positive feces,and the genetic evolution analysis showed that the isolated strain was the prevalent strain in recent years.PoRV and PEDV dual yolk antibodies were prepared,and the challenge protection test of piglets was conducted.The results showed that the immune protection effect of PORV and PEDV was nearly 70%.