The Elementary Research On Screening Marek's Disease Resistance-Related Genes Of Three-Yellow Chicken

Marek's disease (MD) is a lymphoproliferative disease of chicken and has a great impact on poultry industry. Research for screening MD resistance-related genes is beneficial to the breeding of enhancing the genetic resistance in three-Yellow chicken, which is a complementary avenue to vaccination for controlling MD. An elementary research was done and the results were documented in the following:A local MDV named YL040920 was isolated from a Three-Yellow chicken, which had been vaccinated with CVI988 vaccine, with MD visceral tumors by transfected monolayer of duck embryo fibroblast (DEF) with the peripheral blood lymphocytes (PBLs) of the bird. Nine clones of the isolate obtained by the plaque assay were similar in the time of plaque formation and shape of plaque that demonstrated the purity of the isolate. The oncogenicity-related gene meq of the isolate was amplified by polymerase chain reaction (PCR) technique, and then were sequenced and compared with that of the other referent strains of MDV-1. The isolate YL040920 was found to have the characteristic of virulent MDV-1. Specific fluorescent stains localized in the nucleus of the infected DEFs by an immunofluorescence assay (FA) using an anti-MEQ monoclonal antibody 3G12E6. The results demonstrated the isolate was the virulent strain of MDV-1.Day-old chicks of Liangfeng and Huagui were challenged with the isolate and obvious visceral tumors were observed at about 30 days post-infection (PI), showed the acute oncogenicity of the isolate. A developed differential diagnosis technique based on a multiplex PCR for avain neoplastic diseases was used to detect the DNA prepared from the inoculated DEFs and tumor samples of the challenged birds. The characteristic patteins of electrophoresis of the virulent MDV-1 strain were observed from all the samples detected, and free of CVI988 virus, reticuloendotheliosis virus (REV) and avain leucosis virus (ALV).32 day-old chicks of MD resistance line (K group) and 34 day-old chicks of unselective MD susceptible line (Y group) of Xiayan were challenged with YL040920 isolate. The mortality, the percentage of tumor occurance at 12 weeks PI and the timing of death-peak were 91%, 38% and about 5 weeks PI for Kgroup, comparing to those of 97%, 44%, and about 5 weeks PI for Y group. No significant differences were observed between K group and Y group. The excessive virulence of YL040920 isolate was considered to be the main explaintation for the results. Sera were collected respectively from the chickens after the third and the fourth vaccination of Newcastle disease virus (NDV) and detected for antibody against NDV by using hemagglutination inhibition (HI) test. Lower HI titers than normal were observed and provided an evidence of the immunosuppressive condition induced by the infection of virulent MDV-1.A PCR procedure was developed to amplify the fourth intron of growth hormone gene (GH1) from 28 Xiayan chickens challenged with YL040920 isolate, and the PCR products was digested with restriction endonucleidases Sac I and Msp I respectively. No polymorphism was found in the digestion of Sac I , but four genotypes (AA, AB, AC and CC) were classified by Msp I . The genotype frequency of AA, AB, AC and CC were 54%, 15%, 31% and 0% in K group and 93%, 0%, 0% and 7% in Y group. The difference in genotype frequency of GHI between K group and Y group was possibly associated with the resistance to MD, but further research is necessary for confirming this finding.A reverse transcription (RT) PCR procedure was developed to amplify the ala2 domains of B-F locus (B-Fala2) of major histocompatibility complex (MHC) from 6 Xiayan chickens with differences of resistance after the challenge of YL040920 isolate, and then the nucleotides of the amplified B-Fala2 genes were sequenced and compared with the published sequences of haplotypes for B²1, B^21-LIKE, B², B⁶, B⁵, B¹3 and B¹9. The nucleotide substitutions with high variability were observed in the sequences. But the sequences of bird K16 and bird Y30, both showed the highest resistance to M...