Isolation And Identification Of A Marek's Disease Virus And Pathogenicity To Hy-Line Chickens

Marek,s disease,MD is a highly contagious infectious disease caused by Marek's Disease Virus(MDV),which can cause malignant lymphadenopathy in poultry,mainly in the offal Organs,skin and other locations cause tumor lesions.MDV can be divided into three serotypes:serum type 1,pathogenic to chickens and can lead to chicken body tumors,representing the strains have Md5(super intensity)and JW,GA,Beijing 1(virulent);Serum type 2,not on the chicken can not lead to chicken tumor lesions,representing the strains of 301B/1,SB/1,etc.;serum type 3,is a strain isolated from turkeys virus(HVT-FC126 Strain),no pathogenicity to chickens,but clinical trials have shown that chickens can have a better resistance.J subspecies avian leukemia(Avian leukosis subgroup J,ALV-J)was found in the early 1990s by the subgroup of avian leukosis virus(ALV-J)caused by the subprime pulp Cell-like tumor cell proliferation of a tumor disease,but also widespread in the world.In recent years the scope of the disease has changed the host,has spread to the local strains of chickens,causing local strains of chicken occurred in different types of tumors[6],clinically more common bone marrow cell tumor or hemangioma and myeloid cell tumor mixed cases,Indicating that ALV-J pathogenic characteristics and tumorigenicity have changed.In this study,the tumor virus was isolated and identified in the samples and blood samples of the three yellow chicken farms with the outbreak of tumor disease.It was found that the mixed infection of leukemia and Marek's disease was successfully isolated and a strain of MDV was successfully isolated.The main genes were cloned and sequenced,and the pathogenicity experiments were carried out on the purified strains.The results are as follows:1.Isolation and identification of mixed infection of Malik and avian leukemia in three yellow chickensIn this study,according to the clinical and necropsy changes of chicks,the initial diagnosis of tumor disease infection,and the virus isolation and identification.The lymphocytes were isolated from the chickens and the chickens were inoculated with chicken embryos(CEF)for 7 days.The passage was continued for 7 days.Meanwhile,the P27 antigen was detected by ELISA.Cells continue to pass,to observe the presence of plaque.Finally,plaques appeared on the third day of the third generation,and MDV and ALV were identified by indirect immunofluorescence assay(IFA).Among them,monoclonal antibodies BA4 and H19 were positive for the Marek monoclonal antibody.The positive results of the IFA of H19 monoclonal antibody were proved to be the wild strain MDV.The monoclonal antibody JE9 of J subgroup ALV(ALV-J)ALV-J was identified and various results indicated that there were more severe ALV infection in the three yellow flock.The co-infection of MDV and ALV was found by the isolation and IFA identification of the inoculated cells,named GX1701.At the same time,the two genes related to tumor virus were amplified.The results of molecular sequencing confirmed the above IFA conclusion and confirmed the co-infection of avian leukemia and Marek virus.2.Molecular cloning sequence analysis of MDV wild strains in three yellow chickensAt the same time,the genes of the two tumor diseases were amplified,and the sequencing results were consistent with the IFA results.The main pathogenic gene pp38 and meq of the wild-type strain MDV GX1701 were subjected to PCR amplification,molecular cloning and ligation,sequencing analysis.The comparative analysis of the pp38,meq gene and the standard reference strain of the amplified MDV showed that:The homology of this strain was 97.5%and 95.3%,respectively.It was found that the base of the wild-type strain meq gene was CAC at the 575-577 position and had a 177 bp insertion,which was characteristic of the virulent strain.The strain was close to the early isolate of GX0101,and the pp38 gene was More conservative,and GX0101 sequence exactly the same.According to the sequence alignment of the MDV pathogenic genes,the isolates were virulent,but further pathogenicity test is needed for its pathogenicit3.Use of nucleic acid reverse transcriptase inhibitors to remove avian leukemia in MDV wild viruses(AZT)and lamivudine(LAM)were used to eliminate ALV contamination in MDV by nucleoside reverse transcriptase inhibitors.Previous studies have confirmed that the addition of no more than 5 mg/L inhibitor to the culture medium has no effect on the replication and activity of CEF cells.In vitro experiments showed that the addition of 5 mg/L AZT in the culture medium significantly inhibited the replication of ALV on CEF cells.After this intervention,the p27 antigen was negative by ELISA,and the ALV-J monoclonal antibody JE9 for IFA did not find positive cells.So we got the purified MDV GX1701.4.Animal regression experiment and pathogenicity analysis of MDV wildIn order to further observe the pathogenicity of the isolated MDV wild-type strain and whether it could break through the vaccine protection,MDV wild-type strain was infected with 5-day-old sea blue-brown laying hens,and the control group was protected by immunization in one day old Pathogenicity test and immunoprotective experiments.The results showed that the GX1701 strain had a strong virulence,and the clinical symptoms of chickens infected with MDV virus were mainly concentrated in 8-9 weeks.The clinical symptoms were found at 25 days after the first challenge,and the death of chickens first appeared after challenge 10 days,the onset of chicken feathers loose,25 days after the death of the chicks and accompanied by splicing symptoms,infected with GX1701 and immunized CVI988 vaccine sea blue brown chickens first appeared dead chickens after 15 days,and found in 30 days a Cut a tumor dead chicken.Chickens infected with MDV virus alone can achieve 70%and 50%morbidity and mortality,and 10%of the incidence of cancer;immunized CVI988 vaccine sea blue brown chickens can reach 30%and 20%of the incidence and mortality,And 2%of the tumor incidence.Of the natural death chickens infected with MDV virus and chickens that were killed after 14 weeks of attack,the wild virus strains could cause varying degrees of heart,liver and kidney tumors,and individual chickens The glandular stomach of the tumor.;Immunized CVI988 Vaccine of the sea blue brown chickens only one dead chickens were found in the liver when the tumor was found.