Research On Epidemiological Of ALV Of Grandparent, Parent And Commercial Layer Chickens In Several Regions Of Shandong Province And Virus Isolation

ALV-J is found in almost all white feather meat-type chickens in the world in a few years since Payne L.N reported ALV-J in 1988. In recent decades years, ALV-J infection has found in egg-chicken type, even the locality type chicken have covered ALV-J infection. Furthermore, ALV-J infection has appeared new characteristics, firstly, the age infected is ahead of schedule, also the infection range is augment; secondly, the behavior of ALV-J brought are various, the phenomenon about ALV-J infected with other viruses is more and more, and it broutht new problems in preventing and curing. And the third the most serious infection form is haemanqioma ALV-J. Besides it contains the occur of myelocytome. The infected chicken have bloody vesicles on surface skin,joint and follicultis, lastly the chicken died of excessive loss of blood.In order to clarify the prevalence about etiology and serology of the AL in Shandong Province, 1 827samples of serum samples were collected from four regions about Tai'an,Ji'nan,Qingdao and Linyi in Shandong Province, including 597 samples of grandparent layer serum,710 samples of parent layer serum and 520 samples of commercial layer serum;at the same time, 1 209 samples of cotton swabs were collected from these regions above, including 597 samples of grandparent layer swabs,460 samples of parent layer swabs and 172 samples of commercial layer swabs. Serums were tested for ALV-J and ALV-AB antibody and cotton swabs were tested for P27 antigen through ELISA.. The positive rate of grandparent layers antibody of ALV-J is 0 and the positive rate of antibody of ALV-AB is only 0.84%, but the positive rate of antigen of P27 is great in some grandparent layer and this is probably because non-standard crossbreed that result in endogenous virus infectious. The reason of immunological tolerance is either can't be ruled out. The antibody rate of ALV-J is a little higher than ALV-AB. The positive rate of antibody is ascending from grandparent layer to commercial layer. The difference about positive rate of ALV-J and ALV-AB antibody of commercial layer is little in serums among different regions, but there is no obvious ascending of the positive rate of P27 antigen. In this research some suspected haemangiomas type of avian leukosis samples were got from one Hyline brown chinken farm in Shandong Province, and six Subgroup J avian leukosis viruses (ALV-J) were isolated and identified in which two virus strains were isolated from parent and grandparent breeding chickens respectively, and the other four strains from commercial chickens by isolation and identification, IFA, PCR and ELISA. The isolation of the six strains of grandparent,patent and commercial layers is the first time in China. The two virus strains isolated from grandparent and parent breeding chickens were respectly named SDDP1001 and SDDP1002, and the virus strains isolated from commercial breeding chickens were respectly named SDDP1003,SDDP1004,SDDP1005,SDDP1006. The gp85 gene of all the six ALV-J strains were amplified by PCR with a pair of specific primers designed according to the HPRS103 strain. The analysis of sequence of gp85 gene by DNASTAR sofeware showed that Commercial strains SDDP1003,SDDP1004 and SDDP1006 were in the same embranchment with SDDP1002, the rate of homology is between 97.2% and 97.9%, the rate of homology with HPRS103 is between 94.7% and 95.2%; SDDP1005 strain and SDDP1001 strain are in the same embranchment, the rate of homology with HPRS103 is 98.3%. The rate among four commercial strains is 95.0%-99.9%. The rate of homology about six isolated strains and RAV-1 A is only between 12.0%-12.8%; also RAV-2 B. The rate of homology is 12.2%-12.4% with ALV-E. But the six strains have a high homology with JL3-1 and HuB2 (96.2%-99.8%) which isolated in our laboratory. The reason is properly the virus has a variation or they came from other resources through horizontal transmission. All as above implied that ALV-J infected in egg-type groups partially comes from grandparent breeding chickens or parent breeding chickens through vertical transmission, or from other resources through horizon transmission.Infectiong DF-1 cells with the isolated SDDP1001,SDDP1002 and SDDP1006 equivalently, detection form ELISA for p27 antigen indicated that Grandparent SDDP1001 strain replicated the fastest and the result is 10^5.59 TCID₅₀/0.1ml, the result of Parent SDDP1002 strain and Commercial SDDP1006 strain were seemly approximately, the results were 10^4.61 TCID₅₀/0.1ml and 10^4.71 TCID₅₀/0.1ml. The three strains had the similar replication curve. Through the research of same quantity viruses of this three strains infected with DF-1 cells ,the result indicated that the grandparent SDDP1001 strain has a higher infectious virus when equality viruses infected DF-1 cells.ELISA is a frequently used method to wash out the virus infected chickens. There is vendible ELISA in America in 1985, and now in China there is a lack of better ELISA in vendible. The America ELISA is expensive and is inapplicability to large scale detection of farms, so a development of feasible method is needed. Besides cells infected with ALV-J produce no CPE, we can't quantity the virus through TCID50. Yongbaek Kim established a method on detection ALV and ALV-J by real-time PCR, and through comparation about QC-RT-PCR,conventional PCR,Ag-ELISA, the real-time PCR was a very sensitive, accurate method and was easy to perform. The assay was optimized to produce linearity from 10¹ copy/μl～10¹⁰copy/μl in standard curve. It is highly specific and no cross reactivity was detected against other virus. It had a sensitivity of detecting about 10 copies of viral DNA. The assay developed here could provide a powerful tool for quantification of ALV.