Detection Of The Co-infection Of Four Common Vertically Transmitted Viruses In Dead Duck Embryos

In recent years,the intensive degree of duck industry in China has been continuously improved,duck viral infectious diseases are also on the rise year by year,and the co-infection of diseases in large-scale duck farms in China is gradually increasing.The infection of vertically transmitted virus in ducks can not only cause immunosuppression and growth and development obstacles of breeding ducks and meat ducks,but also cause duck embryos to die or hatch weak embryos through vertical transmission of duck embryos,resulting in low hatching rate of duck embryos,which brings great troubles to waterfowl breeding industry,especially breeding duck breeding industry and restricts the healthy development of duck breeding industry.Duck circovirus(DuCV),novel goose parvovirus-related virus(NGPV),novel duck reovirus(NDRV),avian adenovirus(FAdV)pathogens can spread vertically.avian adenovirus pathogens can spread vertically.Therefore,in this study,we used PCR technology to detect DuCV,NGPV,NDRV and FAdV of dead duck embryos collected from hatcheries in different areas of Shandong Province,prepared nucleic acid probes from the detected positive samples,detected the dead duck embryos by dot blot hybridization,recorded the positive rates of different batches,statistically analyzed the co-infection of the sample groups,and compared the results of the two detection methods.On this basis,the positive samples of DuCV and FAdV detected were classified,and the ?C gene sequences of 14 NDRV strains in dead duck embryos were amplified and sequenced,which provided theoretical basis for duck disease prevention and control.This research is divided into four parts:1.Collection of dead duck embryos and detection of four viruses by PCRIn 2021,a total of 308 dead duck embryos will be collected from 8 duck flocks in different areas of Shandong Province,including Jining 1(48),Jining 2(47),Dezhou 1(48),and Dezhou 2(36),Zibo(39),Heze(30),Liaocheng(30)and Linyi(30).DNA and RNA from dead duck embryo tissues were extracted and PCR assays were performed to detect DuCV,NGPV,NDRV and FAdV.The results showed that the individual detection rates of the four viruses in dead duck embryos were 16.56%(DuCV,51/308),18.18%(NGPV,56/308),46.43%(NDRV,143/308),11.36%(FAdV,35/308),respectively.The total detection rates of double infection and triple infection were 19.15%(59/308)and4.55%(14/308),respectively,and no quadruple infection was detected.Virus co-infection was found in all the 8 duck flocks.The detection rates of quadruple infection and triple infection were 62.5%(5/8)and 37.5%(3/8).The test results showed that the co-infection of vertical transmission viruses was common in ducks.Since DuCV and NDRV were immunosuppressive viruses,it was speculated that the co-infection of vertical transmission viruses might be one of the important reasons for the immunosuppression,growth and development disorder,duck embryo death and low hatching rate of ducks.2.Establishment and application of nucleic acid probe detection methods for DuCV,NGPV,NDRV and FAdVIn this experiment,DNA probes were prepared by cloning a conserved sequence of 253 bp,220 bp,138 bp and 307 bp in the conserved region of DuCV,NGPV,NDRV and FAdV gene sequences respectively.The specificity test showed that the prepared DNA probes had high specificity and was negative for hybridization with other pathogenic nucleic acids.The sensitivity test showed that the minimum detection amounts of the DNA probes for DuCV,NGPV,NDRV and FAdV were 11 pg,8pg,10 pg and 10 pg,respectively.The DNA probes were used to detect 308 dead duck embryos from different areas in Shandong.The results showed that the detection rates of DuCV,NGPV,NDRV and FAdV were 11.04%(34/308),12.66%(39/308),42.53%(131/308),and 6.82%(21/308),respectively.The comparison detection results showed that the individual detection rates of the four viruses of DNA probes were lower than those of the corresponding PCR methods.Since the sensitivity of DNA probe detection is lower than that of PCR detection method,it is speculated that the copy number of these four viruses in some dead duck embryos is low.3.Determination and analysis of NDRV ?C gene sequnenceIn order to better understand the relationship between NDRV strains in dead duck embryos and existing strains,the ?C gene sequences of 14 duck embryo-derived NDRV strains were amplified in this study,and phylogenetic tree construction and homology analysis were performed.The results showed that the 14 NDRV strains were in different evolutionary branches from Muscovy duck reovirus(MDRV)and Avian reovirus(ARV).The nucleotide homology among the 14 NDRV isolates was 96.5%-99.6%,with the NDRV ?C gene sequence registered on NCBI was 92.9%-98.3%,and with the NDRV classic strain TH11 was 93.8%-97.2%.The isolates JN-48,JN-50 and JN-61 in this study were most closely related to the TH11 strain,and the homology was 95.3%-97.2%.4.Genotyping of DuCV and FAdVUsing the double PCR method established in our laboratory to detect two genotypes of DuCV,51 DuCV-positive samples were identified.The detection results showed that the detection rates of DuCV-1 and DuCV-2 were 96.08%(49/51)and 9.80%(5/51),respectively.According to the Hexon gene of FAdV,the specific primers for FAdV-4,FAdV-8a,FAdV-8b,and FAdV-11 were designed.All of the 35 FAdV-positive samples were positive for FAdV-4,while no other FAdV serotypes were detected.The results of genotyping showed that DuCV-1 and FAdV-4 were the dominant genotypes in dead duck embryos.In this study,the pathogenic nucleic acids in dead duck embryos were detected by two detection methods,and the ?C gene sequence of NDRV was isolated and amplified,which provided epidemiological basis for the purification of vertical transmission viruses in duck farms.