Establishment And Application Of Indirect ELISA Detection Method For Novel Duck Reovirus

In recent years,an infectious disease characterized by duck spleen necrosis has appeared in China.The sick ducks are depressed and have soft feet.The age of onset of the disease is relatively small,and the disease progresses rapidly,causing necrosis of the spleen,leading to a decline in immunity,and is likely to cause secondary infections of other pathogenic microorganisms,so the mortality rate is relatively high.Novel duck reovirus(NDRV)was considered to be the causative agent of the disease.At present,RT-PCR,fluorescence quantitative PCR and other methods are generally used for the rapid detection of the pathogen for the disease.Compared with those methods,indirect enzyme-linked immunosorbent assay(iELISA)can be used for large-scale serum of the serologic investigation.The ?C protein is the main protective antigen protein of NDRV.In this study,the prokaryotic expression of NDRV ?C protein was used as the coating antigen,and an iELISA method for detecting NDRV serum antibodies was established and initially applied.Part one: Establishment of iELISA for detecting NDRV antibodyThe NDRV strain SD19 was isolated from ducks with necrotic spleen.Primers were designed to amplify the entire gene encoding ?C protein by RT-PCR.The amplified product was ligated into p ET-32a(+)vector and expressed in E.coli BL21.After Western bolt analysis,the expressed protein was used as the coating antigen to establish an iELISA method to detect NDRV antibody in duck serum.The optimal conditions were as follows: the concentration of coated ?C protein was 5 ?g/ m L with 100 ?L/well,the coating time was 12 h at 4 ?,the blocking time was 1.5h at 37 ?,the serum was diluted as 1:80 and incubated 1.5h at 37 ?,the substrate reaction time was 15 min,and the critical value was 0.34.The established iELISA method was utilized to test the single factor serum of GPV,NGPV,MDPV,DHAV-1,DHAV-3 and NDRV negative-positive serum.It was found that all serum values except for the NDRV-positive serum were all less than 0.34,indicating the established the iELISA method has high specificity for detecting NDRV antibody in serum,and the determined cut-off value is appropriate.One hundred duck serum samples were collected and detected by iELISA and the dot blot was also arranged.The results showed that the coincidence rate of the two methods was 93%,indicating that the established iELISA method was feasible for serological investigation of NDRV antibody.Part two: Serological investigation using the established iELISAThe established iELISA was used to detect NDRV antibody in 1000 sera samples of 1 to 40 day old cherry valley ducks from 20 farms in Shandong and Jiangsu provinces.The results showed that the positive group rate of NDRV was 100%(20/20)and the positive individual rate was 53.3%(533/1000);the positive rate of Shandong ducks was 54%(270/500),and the positive rate of Jiangsu ducks was 52.6%(263/500).The positive rates of individual ducks at the age of 1-10 days,10-20 days,20-30 days,and 30-40 days are respectively 58%(145/250),55%(165/300),50.5%(101 /200)and 48.8%(122/250).The iELISA method was also used to detect 500 sera of 14-61-week-old parent-generation Cherry Valley breeding female ducks from 10 farms in Shandong and Jiangsu provinces.The results showed that the positive group rate was 100%(10/10)and the positive idividual rate was 60%(300/500);the positive rate of parent breeding female ducks from Shandong was65.2%(163/250),and the positive rate of parent breeding female ducks from Jiangsu was54.8%(137/250).Additional,the positive rates of parent breeding female ducks at 14-25 weeks,26-35 weeks,36-45 weeks,and 46-61 weeks were 61%(122/200),53%(53/100),65%(65/100),60%(60/100).The detection rate of male ducks in an ancestral duck breeding farm in Jiangsu Province was 96%(48/50).The above serological testing of cherry valley duck ancestor breeding male ducks,parent breeding female ducks and commercial meat ducks showed that NDRV has been widespread in duck flocks in Shandong and Jiangsu provinces,and we should pay more attention to its infection.