Preliminary Study On The Etiology Of Cherry Valley Duck With Feathers Shedding Syndrome

Since 2017,a clinical disease,feathers shedding syndrome(FSS),with part or all of the body feathers falling off and the feather roots difficult to be removed after slaughter(burr ducks),has occurred in cherry valley ducks in the three provinces(Shandong,Anhui and Jiangsu)of eastern China.The disease is particularly serious in 2019 and mainly occurred in Cherry Valley meat ducks.The incidence of disease is 3%-70%.The diseased ducks are mainly characterized by stunted growth,lower feed intake,and more and more feather loss;some ducks will die,and the mortality rate can reach 40%,causing huge losses to meat duck farmers.Duck circovirus(DuCV)and novel goose parvovirus-related virus(NGPV)infection may cause similar clinical symptoms.In this study,we established a triplex-PCR method for the simultaneous detection of DuCV-1,DuCV-2,and NGPV.The feather sacs and various organs of ducks with FSS were collected in Shandong,Anhui and Jiangsu provinces and detected by the triplex-PCR method.Based on this,whole genome of six DuCV strains and six NGPV strains in clinical disease materials were amplified and the sequences were analyzed,and animal experiment was did to explore the causes of the disease.These results were expect to provide important information for the scientific prevention and control of FSS disease in the duck industry.This research is divided into four parts:1.Establishment of a triplex-PCR for detection of DuCV-1,DuCV-2 and NGPVBased on the conserved sequences of DuCV-1,DuCV-2 and NGPV,five primers were designed using Oligo7.0 software,and a triplex-PCR method was developed to detect DuCV-1,DuCV-2 and NGPV in one step.This method was very specific for detecting the three viruses DuCV-1,DuCV-2 and NGPV in one step,without reaction to goose parvovirus(GPV),muscovy duck parvovirus(MDPV),duck fever virus(DPV),duck hepatitis B virus(DHBV),E.coli(E.coli),Riemerella anatipestifer(RA),and DHAV and other pathogens.The minimum detection concentration for DuCV-1 is 50 copies/?L,for DuCV-2 is 50 copies/?L,and for NGPV is 5×10~3copies/?L,respectively.2.Investigation and analysis of FSS duck etiological situationIn order to better understand the prevalence of Cherry Valley Meat Duck with FSS,a total of 540 feather sacs(twenty samples in every farm)and 540 samples of various organs(60samples of each organ)were collected from 27 duck flocks with FSS in Shandong,Anhui and Jiangsu provinces from August 2018 to June 2019,and DuCV-1,DuCV-2 and NGPV were tested with the established triplex-PCR method.The detection rate of DuCV in the feather sac was 78.89%(426/540)and NGPV was 82.78%(447/540),the co-infection was 70.00%(378/540).It was found that co-infection was more serious in ducks at 4-5 weeks of age.DuCV and NGPV were detected in all organs,and the highest detection rates of the two viruses were in the heart with 88.33%(53/60)and 84.91%(51/60)respectively,while the lowest detection rates of the two viruses were in the brain with 21.67%(13/60)and 15%(9/60)respectively.DuCV viral load in the FB was the highest,and the NGPV viral load in the heart was the highest.The viral loads of DuCV and NGPV in the feather sacs were higher than tht of other organs,and the viral loads of DuCV and NGPV in lungs were lowest.According to our clinical test results,the distribution of DuCV and NGPV in FSS diseased ducks was simlar,and the two viruses had same tissue tropism.3.Whole genome sequencing and analysis of DuCV and NGPVWe designed corresponding primers based on the entire genome sequence of DuCV and NGPV registered on the NCBI to perform genome-wide amplification of 6 DuCV stains(three each of DuCV-1 and DuCV-2)and 6 NGPV strains.Meanwhile,the evolution tree was construct with Megline 7.0 software and the main proteins and genes were compared and analyzed.The results show that the 6 amplified DuCV sequences were not significantly different from other uploaded DuCV sequences in NCBI.The homology of DuCV-1 was93.4%to 98.5%,and the homology of DuCV-2 was 96.9%to 98.7%.The 6 NGPV strains were located in the same small branch,with 98.1%to 98.8%homology with other NGPV strains and 91.0%to 95.6%homology with GPV strains.Sequence analysis results revealed that DuCV Rep protein had no common mutation site,while DuCV-1 had only one common mutation site in Cap protein,and DuCV-2 had 6 common mutation sites in Cap protein.To NGPV,there were 6 common nucleotide mutations,including two increased bases in the ITR region,and 7 and 12 nucleotide mutations causing amino acid mutations in the Rep protein and Cap protein,respectively.At the same time,the corresponding secondary structure and tertiary structure of the VP3 protein in Cap protein were predicted,and some amino acid variation sites were further analyzed.4.Animal challenge experimentsIn this study,animal challenge experiments were conducted to verify the relationship between FSS disease and DuCV and NGPV.A total of 166 1-day-old healthy Cherry Valley ducklings(detected without DuCV and NGPV and other pathogens)were randomly divided into four groups:control group(16 ones),NGPV single infection group,DuCV single infection group and NGPV and DuCV co-infection group(50 ones per group).At 7 DPI,14DPI,21 DPI,28 DPI,35 DPI and 42 DPI,8 ducks were randomly selected from each group,and internal organs and feather sacs were collected for virus load comparison.At each period,the feather sacs were collected separately from 16 ducks to record the positive rate of the viruses.At the same time,three ducks that tested positive for the feathers sac were tracked to analyze the dynamic changes of the virus in the feathers sac at different periods.The study found that the clinical symptoms of defecation were the most serious in the ducks of the co-infection group.The presence of the respective virus could be detected in the feather sac at different time points,and the positive rate of the virus in the feather sac increased with increasing of age,the infection rate of DuCV and NGPV was higher in the co-infected group than in the single infection group,and the viral load was higher than that of the single infection,reaching its peak at 21 DPI.The results showed that DuCV and NGPV had similar tissue tropism and replication law in the internal organs and feather sac of challenged ducks.Through comprehensive analysis of clinical test data and animal challenge test data,this study preliminarily concluded that the co-infection of DuCV and NGPV may cause FSS in ducks.