Establishment And Application Of Indirect ELISA For Detection Of Serum Antibody To Subtype C Avian Metapneumovirus In Ducks

Avian metapneumovirus(a MPV)infects turkeys,chickens,ducks and some wild birds,causing acute upper respiratory tract infection,accompanied by decreased egg laying rate and other symptoms.It is highly infectious and spreads rapidly,which is easy to cause secondary infection and high mortality.The disease is widespread in the poultry industry and has caused serious economic losses in many countries all over the world.a MPV was first isolated from chickens in China in 1999,and epidemiological investigation showed that the virus was widely infected in chickens in China.In 2014,the subtype C a MPV(a MPV/C)was isolated from Muscovy ducks for the first time in China.Until now,there are few studies on duck-derived a MPV.In addition,the virus is not easy to isolate,and the harm to the breeding industry is increasing.Therefore,it is particularly important to establish a rapid and accurate diagnosis method.there are few studies on ducks.Indirect enzyme linked immunosorbent assay(i ELISA)has the advantages of sensitivity,rapidity,simplicity and cost effectiveness.It can be applied to large-scale serological investigation of the disease and provides a technical means for further understanding the situation of a MPV infection in domestic ducks.This study is divided into the following two parts:Part one:Isolation and identification of LY0913 strain a MPV/CIn this study,primers were designed according to the conserved sequence of a MPV Fusion(F)gene published in Gen Bank to detect the clinically suspected a MPV infected materials by RT-PCR.The detection results showed that the target fragment of 336 bp was specifically amplified.The positive material was ground first,then the supernatant was taken,and the duck embryo was inoculated by yolk sac.After blind transmission to the fifth generation,the pathological changes of duck embryo were obvious.Then take the positive allantoic fluid and culture it on Vero cells.Syncytia appeared 72 hours after challenge.The allantoic fluid and the supernatant of diseased cells were collected for RT-PCR detection,and then the whole gene of F protein was amplified for sequencing.MEGA 11,Meg Align and other software were used to analyze the homology and construct the evolutionary tree.The final genotyping results showed that it had the highest homology with a MPV/C nucleotide,up to 94.6%-99.8%;The homology with a MPV/A and a MPV/B was low,69.9%-70.0% and67.5%-73.4% respectively.Therefore,the isolate was a MPV/C and named a MPV/C LY0913.This is the first time that a MPV/C was isolated from Cherry Valley duck.Part two:Establishment and application of indirect ELISA for detecting a MPV/C serum antibodyAs the main antigen structural protein of a MPV,F protein was selected as the diagnostic antigen to establish an i ELISA method for detecting a MPV/C serum antibody.Firstly,the recombinant plasmid p ET-32a(+)-F was successfully constructed and transformed into E.coli BL21.The F protein was of was expressed successfully by IPTG induction.After purification and Western bolt analysis,the expressed protein used as coating antigen to establish the i ELISA method.The optimum conditions after optimization: the antigen concentration was8?g/m L,the coating conditions were 4? overnight,the serum dilution was 1:100,the sealing fluid is 1.5% BSA,closed at 37? for 2h,the serum reaction time was 90 min,the dilution of enzyme labeled secondary antibody was 1:4000,the substrate reaction time was 15 min,and the cut-off value is 0.238.The specificity test and dot blot method were used to detect the established method.The results showed that the detection values of the positive sera of NDRV,Du CV,DHAV-1,DHAV-3 and NGPV were less than 0.238,whereas the value of the positive a MPV/C serum was 0.614,and the coincidence rate of the i ELISA and dot blot detection was 91%,indicating that the established i ELISA method has specificity and applicability.Part three:Serological investigation of a MPV/CThe established i ELISA method was used to detect the serum of 450 Cherry Valley meat ducks from 11 farms in Shandong and Jiangsu provinces.The results showed that the positive rate of a MPV/C antibody was 33.3%(150/450),among which the positive rate of Shandong meat duck individuals was 33.6%(84/250),and the positive rate of Jiangsu meat ducks individuals was 33.0%(66/200).The established i ELISA method was also used to detect the serum of 640 parental breeding ducks from 11 farms in Shandong and Jiangsu provinces.The results showed that the positive rate of a MPV/C antibody was 50.9%(326/640),among which the positive rate of Shandong duck individuals was 55.9%(179/320),and the positive rate of Jiangsu duck individuals was 45.9%(147/320).A total of 1090 serum samples of Cherry Valley ducks from 25 farms were detected by this method.The total positive rate of a MPV/C antibody was 48.9%(533/1090),and the group positive rate was 72.0%(18/25).The group positive rates of meat ducks and breeding ducks were 63.6%(7/11)and 78.6%(11/14)respectively.According to the above serological results,a MPV/C infection was widespread in duck populations in parts of Shandong and Jiangsu provinces,which should be paid great attention to.