Protective Efficacy Of Vaccinal Immunity To Marek's Disease By Congenital Infection With Subgroup J Avian Leukosis Virus And Molecular Identification Of Different Marek's Disease Vaccine Strains

Marek's disease?MD?is a malignant lymphoproliferative disease of chickens,which is caused by Marek's disease virus?MDV?.Vaccination is the most effective measure to prevent MD?However,CVI988/Rispens is the most widely vaccine has been used in the recent years.Marek's disease occurs frequently in chickens after immunization with CVI988/Rispens vaccine,which has caused significant economic losses in the poultry industry.Avian leukosis virus?ALV?is an immunosuppressive pathogen of chickens that associated with a variety of neoplastic diseases,it is mainly transmitted through vertical infection.Epidemiological investigation revealed that MDV-infected flocks are often co-infected with ALV and other immunosuppressive pathogens,The congenital infection with ALV plays an important role for the incidence of MD after CVI988/Rispense vaccination.To confirm this deduction,we investigated congenital infection of J subgroup ALV on the protection effect of CVI988/Rispens vaccine through a series of animal experiments.In addition,according to lacking specific identification methods for the meq-deleted marek's disease vaccine strain SC9-1 which was developed in our laboratory and MDV wild strains and other vaccine strains,the molecular identification assays were established to distinguish the SC9-1 strain from other vaccine strains of MD.1.Protective efficacy of vaccinal immunity to marek's disease by congenital infection with subgroup J avian leukosis virusTo study the congenital infection of J subgroup ALV on the protective efficacy of CVI988/Rispens vaccine,200 SPF eggs were divided into 5 groups with 40 eggs per group.Group 1and 2 were congenital infections of ALV.At the age of 6 embryos,J subgroup ALV NX0101 strain was inoculated with dosage of 500TCID₅₀0 via the yolk sac route.After hatching,the chickens from groups 1,2,3 and 5 were subcutaneously vaccinated with CVI988/Rispens.The chickens from groups 2,3,and 4 were infected with virulent MDV GX0101 at a dose of 2000 PFU at 6 days of age by intra-abdominal injection.All chickens of each group were observed daily throughout the entire experimental period.Necropsy was performed on all chickens that died or were euthanatized.Suspicious lesions were collected for histopathologic confirmation.The body weight of each group was weighed on the 1st,2nd,3rd,4th,5th,and 6th weeks after the MDV GX0101 infection.After immunization against Avian Influenza virus?AIV–H9?and Newcastle disease virus?NDV?inactivated vaccines,all groups were detected antibody titers at weeks 3,4 and 5.After 7dpi,5 chickens were randomly selected from each group to evaluate the immune organ indices of thymus and bursa.At 5,10,14,21,and 28 days of post infection?dpi?with GX0101,6 chickens in groups 2,3and 4 were randomly selected to collect anticoagulant blood lymphocytes for DNA extraction,the index of copy number of GX0101 were detected using fluorescence quantitative PCR.The results showed that congenital infection with ALV-J increased the mortality rate of CVI988/Rispens-immune GX0101-challenged chickens,the mortality rates of chickens in groups 1,2,3,4,and 5 were 5.7%,20%,2.9%,37.1%and 0%,respectively.Congenital infection with ALV-J and CVI988/Rispens-immune GX0101-challenged group significantly reduced the body weight,and caused atrophy of the thymus and bursa of Fabricius,and also significantly reduced antibody levels in inactivated vaccines of AIV-H9 and NDV,increased the replication of GX0101,comparing with CVI988/Rispens-immune GX0101-challenged group.Therefore,congenital infection with ALV-J reduced the protective efficacy of the MD vaccine and increased the replication of GX0101 and pathogenicity,the results may indicted that congenital infection with ALV is one of the reasons for the increasing incidence of MD in vaccinated chickens.2.Molecular identification of different marek's disease vaccine strainsIn order to rapidly distinguish meq-deleted Marek's disease vaccine strain SC9-1 and other commercial vaccine strains?CVI988/Rispens,814,HVT Fc-126?,according to the viral genomic sequences of SC9-1 and other commercialized MDV vaccine strains,the methods of PCR and DNA blot hybridization were establish,three pairs of primers named F1/R1,F2/R2,and F3/R3 were designed and the probes labeled with digoxin specific for the serotype-I MDV meq gene and SC9-1 unique REV-LTR fragment were synthesized.The results showed that 184-,1297-and 1297-bp products were amplified using SC9-1,CVI988/Rispens,and 814viral DNA as templates employing primer F1/R1,but no specific band was obtained using HVT Fc-126 viral DNA.Also,a 746-bp product could be amplified when using CVI988/Rispens and 814 viral DNA as templates employing primer F2/R2,but no specific bands were obtained using SC9-1 and HVT Fc-126 viral DNA.In addition,a 512-bp product could be amplified when using SC9-1 viral DNA as a template employing primer F3/R3,but no specific bands were obtained using the CVI988/Rispens strain,814 and HVT Fc-126 viral DNA.A dot-blot hybridization assay was also established.The CVI988/Rispens and 814 viral DNA showed a positive by using a probe specific for the meq gene,at the same time,SC9-1and HVT Fc-126 viral DNA showed a negative.When using a probe specific for the REV-LTR fragment,SC9-1 viral DNA demonstrated a positive result whereas CVI988/Rispens,814 and HVT Fc-126 viral DNA showed a negative.In conclusion,we established methods of PCR amplification and nucleic dot-blot hybridization that could be used for rapid distinction of SC9-1 and other commercial vaccines.