Epidemiological Investigation Of Avian Leukosis In Chinese Local Chicken Breeds And Research On Detection And Eradication Technology Of Exogenous ALV

Avian leukosis is the general designation for a variety of tumors induced by avian leukosis virus(ALV)/sarcoma virus.Exogenous ALVs are classified as subgroup A,B,C,D,J,and K according to the antigenicity of virus envelope protein.Avian leukosis was reported in the worldwide scale since the first isolation of ALV in 1908,and the viruses were gaining a stronger foot-hold in broilers,layers,or local breeds in China.Besides typical neoplastic lesion,ALV infection can cause significant economic losses due to immunosuppression,stunted growth,laying drop.ALV can spread vertically in chickens,there is no effective treatment and available vaccine for avian leukosis.Pathogen detection,culling positive chicken to accomplish the purpose of eradication are the most effective precautionary measures.The international breeding corporations were success in the eradication of exogenous ALV in later 1980s,and the eradication of ALV-J was realized in early 2000s.Some breeding companies have carried out eradication program in core breeder group since 2002 in China,and several have achieved outstanding effects.Avian leukosis remains the prevalent feature in yellow-feather chickens and the majority of local breeds to pose severe threaten on poultry genetic resources.The main contents of the paper were included:Epidemiological investigation of avian leukosis in 26 local breeds to acquire the dynamics of avian leukosis in local breeds.Systematic analysis of the genome of an isolate of ALV-K to obtain information about the origin and molecular characteristics.Establishment of a multiple PCR to detect ALV-A,ALV-B,ALV-J and ALV-K circled in local breeds at the broadest level.Feasibility analysis of the detection method for ALV in semen of breeder was also contacted.The eradication program in core groups of 4 local breeds(DX,LY,XJ,LH)was provided an experience for chicken farm.1.Epidemiological Investigation of Avian Leukosis in local Chicken BreedsTo understand the epidemiology of avian leukosis in local chicken breeds in China,an investigation was carried out in 26 breeds of stock farm from 2013 to 2017.We detected sera antibody against ALV-J and ALV-A/B,p27 antigen in albumen,virus in plasma and tissue samples.We also cloned and sequenced env gene of exogenous ALV,and anlyzed phylogenetic analysis of gp85 sequences among the isolates with different subgroup reference strains from GenBank.The results of serological investigation showed that the ALV-J antibody was positive in 22 local breeds.ALV-J and ALV-A/B antibody were both positive in 18 local breeds.ALV-J antibody positive rate in most of breeds was higher than that of ALV-A/B antibody,and several breeds were over 50%.It’s showed that there were shedding-virus from hens in all breeds.Notably,p27 antigen positive rates in YJ,WX,BJ,DJ and ZJ breeds were over 50%.The results of virus isolation showed that all breeds were positive to ALV but the infection levels were significant different among breeds.The viremia rates of hens in the majority were higher than those of roosters(15/21),but not significant different among them.Of 56 isolates of exogenous ALVs,27 strains were belonged to ALV-J,17 strains were belonged to ALV-K,8 strains were belonged to ALV-B,3 strains were belonged to ALV-A,and 1 strain was belonged to ALV-C.There were 9 stains of ALV-J,3 strains of ALV-K,and 1 strain of ALV-A isolated from tissue samples.Besides ALV-J,ALV-K was the most serious threaten for local chicken breeds.Point mutation,insertion and deletion variation were common and partially regular in gp85 sequences of ALV isolates.It’s indicated the universal and complexity of ALV infection in local chicken breeds and eradication of ALV.2.Whole Genome Sequencing and Analysis of ALV Subgroup KTo identify the characteristics of ALV-K genome,one isolate of ALV-K(JS13LY19)from plasma of LY breed was used for cloning and sequencing of proviral DNA.The gene fragments were compared with the reference strains from GenBank.The genome of JS13LY19 was 7483 bp and was coincided with retrovirus type C,lack of oncogene.The identities of gp85 sequence of the isolate with ALV-K reference strains were 90.9%to 98.6%and were significantly higher than those with the other subgroups.The most identity of JS13LY19 was that of JS11C1 strain.There were regular variations in multiple sites and different changes in 5 sites in the meanwhile.LTR,UTR,gag,pol,gp37 of JS13LY19 had higher identity with endogenous ALV,which was 92.0%to 99.4%.LTR U3 region of JS13LY19 missed a CAAT enhencer box,a PRE box,a CArG box,and a Y box.It’s speculated that JS13LY19 was generated by the recombination of JS11C1 and endogenous ALV.Endogenous ALV LTR and partial deletion of transcriptional regulatory elements in U3 regions may give rise to imperfect transcription ability and weaken in the pathogenicity of JS13LY19.The studies on the biological characteristics were necessary in the further.3.Establishment of Multiple PCR for ALV Detection and Its Preliminary ApplicationEpidemiological investigation showed that there were exogenous ALVs prevalent in local chicken breeds such as ALV-J,ALV-K,ALV-A,or ALV-B.One pair of universal primer PF for the above ALVs and four pairs of special primers(AR,BR,JR,KR)were designed to detect the infection situation of chicken groups.The standard plasmid was constructed,and primer concentration,annealing temperature,cycle number were optimized to establish a multiple PCR for detection of ALV-J,ALV-K,ALV-A,ALV-B at the same time.The specificity,sensitivity and repeatability of multiple PCR were tested with 119 samples from local breeds.The optimized conditions of the multiple PCR were 0.2μM PF,0.1 μM AR,0.1 μM BR,0.1 μM JR,0.1 μM KR,with 56.0℃ of annealing temperature and 30 cycles.The limit of quantification of DNA was 10 pg/μL,and the established multiple PCR had high specificity and good repeatability.The result of 119 ALV samples detected by the multiple PCR was coincided with epidemiological investigation.Therefore,the multiple PCR could be more effective detection for multiple infection of ALV.In addition to single infection of ALV in local chicken breeds,dual infection such as ALV-B+J,ALV-J+K,and triple infection such as ALV-A+B+J,ALV-A+B+K,ALV-B+J+K were found in multiple PCR detection.The multiple PCR could provide technical support for preliminary identification of ALV4.Comparison and Preliminary Application of Detection Methods for ALV in Breeder Rooster’s SemenThe objective of this study was to evaluate the feasibility and practicality of detection for ALV in breeder rooster’s semen,and hoped that it would be helpful for the breeders to make the ALV eradication program.Twenty-three semen samples were collected form LK breeder roosters and were detected by 6 kinds of different methods.Then the selected methods were applied to detect ALV in 551 breeder roosters which were from LK,MQ and LH local breeds.As a result,the positive coincidence rate of isolation of exogenous ALV was the highest when semen samples were centrifuged and then diluted at 1:28 to 1:56,compared with plasma samples.The preliminary application results showed that the positive rates of p27 antigen by ELISA for semen samples from different breeds were all the highest,but could not select out all the positive roosters by isolation of exogenous ALV in semen and plasma samples,since there were false negative.The isolation rates of exogenous ALV in plasma and semen samples collected from the same breed were different,their positive coincidence rates were both lower than 50%in LK and MQ breeds,so the two methods could not replace each other.The isolation rate of exogenous ALV in semen samples from LH breed was significantly higher than that of plasma samples,and was also higher than the positive rate of exogenous ALV in plasma samples detected by Dot-blot,their positive coincidence rates were 100%and 36.4%.The experiment showed that it was feasible and necessary to detect ALV in breeder rooster’s semen.As eradication process and infection status in different breeds were different,detection methods should be flexibly applied.5.Demonstration Application of Eradication of Avian Leukosis Virus in Local Chicken BreedsThe ALV eradication program was carried out in the core stocks of four local chicken breeds(DX,LY,XJ,LH)in the farm from 2013 to 2016.The serial measurement included that building of new chicken house,detection of p27 antigen in excrement of 1-day-age chick,virus isolation from plasma at 6 weeks of age,virus isolation from plasma or semen and detection p27 antigen in albumen at 25 to 27 and 36 to 40 weeks of age.On the base of effective management,the obvious progress in four breeds after 2 to 3 generations was achieved.For DX breed after 3-generation eradication,the positive rate of p27 antigen in albumen was reached from 26.4%to 0.3%at 25 to 27 weeks of age.The positive rate of virus isolation from plasma went from 36.4%to 0.4%at 36 to 40 weeks of age.Virus isolation rate was zero in rooster in two successive generations.For LY breed after 3-generation eradication,the positive rate of virus isolation from plasma went from 33.2%to 1.2%at 6 weeks of age,and the positive rate of p27 antigen in albumen and the positive rate of virus isolation from plasma at 36 to 40 weeks of age declined to zero.For XJ breed after 2-generation eradication,the positive rate of virus isolation from plasma went from 61.8%to 0.9%.For LH breed after 2-generation eradication,the positive rate of p27 antigen in albumen declined from 20.7%to 1.9%at 36 to 40 weeks of age,and the positive rate of virus isolation from plasma was down to 0.2%.The effect of ALV eradication on the production performance of breeds,after 3-generation eradication,the mortality rate of DX breed in the brooding,breeding and laying period decreased.The effect in the first generation was the most significant.The number of eggs laid at 43 weeks increased about 13,and the number of eggs laid at 66 weeks increased about 23.The established eradication program could be used for the small group of local chicken breeds,and it’s a good practice for other stock farms.