Differences In The Efficacy Of Duck Tembusu Virus Recombinant Adenovirus Vaccine(rADV-E-IL-2 Strain) Immunized With Different Immunization Methods

Duck tembusu virus(DTMUV)has caused serious economic losses to the current waterfowl farming industry.Vaccination is the key to control the disease.Besides the immunogenicity of the vaccine itself,the immune pathway of the vaccine was extremely important for the immune effect.This experiment immunized the ducks with different immunization methods for the recombinant vector vaccine(r ADV-E-IL-2)that has been successfully constructed in the laboratory with adenovirus as the carrier,DTMUV E protein as the antigen,and duck IL-2 as the adjuvant,and discuss the immune rules of ducks with different immunization methods.In this study,one-day-old ducklings(n=170)were randomly divided into 5 groups in each group.The first immunization was performed at 7-day-old and boosted at 12-day intervals.The r ADV-E-IL-2 vaccine was immunized with the same dose(10~7 PFU)by the following immunization protocol:both the first immunization and the booster immunization were immunized by intramuscular injection(i.m.);immunized by intramuscular injection firstly and boosted by eyedrop and nasal inoculation(i.m+e.n.);firstly immunized and boosted by eyedrop and nasal inoculation(e.n.);while establishing positive group(intramuscular WF100 vaccine)and negative group(intramuscular injection of PBS).The serum,spleen and duodenal detection antibodies and cytokine changes were collected to detect the antibody levels and cytokines at 3 dpi,10 dpi,17 dpi,24 dpi,31 dpi,38 dpi,48 dpi,and 60 dpi,respectively.10 ducks were randomly selected to attack with 10~5PFU of DTMUV at 24dpi.All ducks were euthanized at 31 dpi,and the indicators of challenge test were tested.The results showed that the inoculation of r ADV-E-IL-2 vaccine by the three immunization protocols can effectively stimulate the body to produce high levels of neutralizing antibodies and specific Ig G antibodies.However,there was no significant difference in Ig G produced in group i.m.,group i.m.+e.n.and group e.n..The first immunization,the neutralizing antibody level in the i.m.+e.n.Group(1:68)was significantly higher than that in the i.m.group(1:40)(p<0.05)at 38 dpi.The specific antibody Ig A content in the duodenum of the group i.m.was significantly lower than the group i.m.+e.n.and the group e.n.(p<0.05)at 17-60 dpi.The first immunization,the expression of IL-10 in the spleen of the i.m.+e.n.group(4.61±0.45)and e.n group(4.90±0.15)was significantly higher than in i.m.group(3.49±0.47)(p<0.05)at 17 dpi..The expression of IL-4 in spleen at 24dpi,the i.m.+e.n.group(6.93±0.56)was significantly higher than that in the e.n.group(7.97±0.15)(p<0.05).The expression of IFN-γin spleen at 24dpi and 31dpi,the i.m.+e.n.group(9.26±0.28)(11.45±0.89)were significantly higher than that in the i.m.group(8.09±0.34)(9.65±0.46)(p<0.05),and the i.m.+e.n.group(9.02±0.26)was significantly higher than that in the e.n.group(8.19±0.23)(p<0.05)at 60 dpi.In the challenge protection test,there was no morbidity or mortality in each group except in the negative PBS control group.At autopsy,the i.m.group,i.m.+e.n.group,e.n.group and PC i.m.group all presented different degrees of splenomegaly and liver bleeding spots.The copy number of DTMUV in the heart of group i.m.+e.n.(5.92±0.19copies/g)was significantly lower than of group i.m.(6.32±0.16 copies/g)(p<0.05).The copy number of DTMUV in the liver of e.n.group(5.68±0.09 copies/g)was significantly lower than that of i.m.group(6.23±0.28 copies/g)(p<0.05).And the copy number of DTMUV in the brain of group i.m.+e.n.(5.58±0.17 copies/g)was significantly lower than that of group i.m.(5.99±0.17 copies/g)and group e.n.(5.97±0.62 copies/g)(p<0.05).HE staining pathological sections of heart,liver,spleen,kidney and brain tissues of each group showed different degrees of lesions.The liver lesions in group i.m.had vacuoles of various sizes in the visual field,and hepatic cord disorders and hepatic lobular structures were not obvious.Heart edema appeared in group e.n..Granular degeneration and interstitial lymphocytes appeared in renal tubular cells in groups i.m.,i.m.+e.n.,e.n.and group i.m.,perivascular inflammatory cells increased.This study confirmed that the r ADV-E-IL-2 vaccine can effectively stimulate the body to produce good immune response through the first intramuscular injection of intramuscular injection,effectively reduce the virus content in various tissues,produce slight pathological damage,and better protect the body from DTMUV.