Isolation And Identification Of Three Marek’s Disease Virus Field Strains And Analysis Of Pathogenicity

Marek,s disease(MD)is a highly contagious lymphoproliferative disease of domestic chickens induced by Marek,s disease virus(MDV),which characterized by a mononuclear celluar infiltration of the peripheral nerves,bursa and thymus atrophy,viscera,muscles and skin.In recent years,MDV infection will occur and cause tumor even vaccinated with CVI988/Rispense to the chicken flocks,MD outbreaks could also be an important economic problem in the poultry industry In this study,three MDV wild strains were isolated and identified from different broiler chicken flocks after vaccine with CVI988/Rispense,the main pathogenic genes were cloned and sequenced,reactivity of purified strains were tested by Hy-Line Brown chickens.Isolation and identification of three MDV field strains and sequence analysis of pathogenic gene.1.Isolation and identification of three Marek’s disease virus field strains and sequence comparison of genes involved in pathogenesisIn order to study the prevalence and distribution,virulent variation of MDV in china,three isolates of chicken embryos were isolated from lymphocytes of broiler chicken flocks that had been vaccinated with CVI988/Rispense by clinical necrospy,virus isolation,IFA assay and PCR amplification were carried out.Three field isolates named as SDAU1502,SDAU1502,SDAU1503,respectively.The main pathogenic genes VIL8,pp38 and meq of MDVs were amplified by PCR compared with the standard reference strains for amino acid sequence analysis.Phylogenetic analysis showed that the homology of meq gene of SDAU1503 strains was 99.1% with CVI988/Rispens,and the homology of SDAU1502 is100% with SDAU1501;pp38 gene is relatively conservative,the homology of SDAU1502 is99.7% with GX0101,99.3% homological to other foreign reference strains;the homology of vIL8 gene of SDAU1503 is 99.3% with foreign standard starins,the homology of SDAU1501 and SDAU1502 were close to GX0101,98.5% and 99.3% respectively,and showed 97%homological to foreign reference strains.According to the phylogenetic analysis with the pathogenic genes for three isolates,we found that the pathogenic genes of SDAU1501 and SDAU1502 were consistent with virulent strains,SDAU1503 is consistent with attenuated vaccine strain.Due to multiple mutations in different pathogenic genes of three isolates,we could infer that three field strains are virulent and attenuation strains respectively,but the pathogenicity of three isolates requires further study to determine their pathogenicity.2.Analysis of pathogenicity of three Marek’s disease virus field strainsIn order to further observe the pathogenicity of three Marek’s disease virus field strains,we conducted on pathogenic experiment for three isolates and inoculated to one day old Hy-Line Brown chickens.The results showed that SDAU1501 and SDAU1502 strains were highly virulent.The chickens showing the clinical symptoms at 21 days post infection with SDAU1501 and SDAU1502.The first death of chickens occurred at 25 days post inoculation,the incidence of chicken shows feathers disorder,and appeared clinical symptoms focused on 7 to 10 week.The chickens infected with SDAU1501 have morbidity and mortality rates were 100% and 90%,the percentage of tumor index was54.The chickens infected with SDAU1502 showed 100% morbidity,77% mortality and the percentage of tumor index was 44..SDAU1503 strain is less virulent,the chickens infected with SDAU1503 appeared clinical symptoms focused on 10 to 12 week post inoculation,the clinical symptoms were found at 47 day post infection at the earliest and the death appeared at52 day post infection caused 70% morbidity,34% mortality and 14% Oncogenicity.The chickens were dead due to the infected with three strains and killed chickens after 16 weeks post infection were necropsy and postmortem examination,the tumors could be found in heart,liver and kidney organs,and individual chicken appeared tumors in skin,glandular stomach,and ovarian.The chicken infected with SDAU1501 and SDAU1502 showed tumors in heart,liver and kidney with the size of a pea cardiac at 6-8 week post inoculation,the dead chicken began to appear diffuse liver tumor at 10-12 weeks after infection,the dead chicken occur in glandular stomach tumor lesions at 14-16 weeks after infection.However,the chickens infected with SDAU1503 had a slight delay of tumor development,but the liver lesions occurred was earlier than the pathological changes of the heart organ,kidney and liver tumors occurred at 8 week post inoculation and cardiac tumor occurred at 14-16 week after post inoculation.This study demonstrates that new variations strains of MDV is occurring in recent years,they became more virulent,The current vaccine have been unable to control the epidemic situation.Therefore,prevalence and genetic monitoring of MD should be processed,Meanwhile,more attentions should be given to MDV vaccine development.