| Chicken major histocompatibility complex (MHC) gene is of great interest to poultry breeding scientists for its extraordinary polymorphism and many studies confirm that MHC haplotypes shown significant difference to the AL resistance. Previously, the chickens of BWEL-SPF were genotyped for four microsatellites located within MHC gene, and the homozygous individuals were selected and developed to three inbred lines named as G1, G5 and G6 with specific MHC haplotypes. The aim of this paper was further study the resistance difference on G1, G5, G6 lines to AL and finally established a more resistant and susceptible inbred lines.In order to remove the ALV-infected chickens from the whole BWEL chicken line, the dot-blotting hybridization assay has been established based on the group special p27 antigen of ALV. After that, three specific pairs of primers were designed based on the p27 and env gene of ALV A subgroup and env gene of ALV E subgroup. A triplex PCR assay used for detecting ALV and differentiated exogenous and endogenous ALV was developed to compare the detective results of dotblot assay. A duplex quantitative real-time reverse transcription polymerase chain (qRT-PCR) method to detect and quantify the virus load of avian leukosis virus subgroup A and B (ALVA/B) was also developed.The assay was specific for ALVA/B and was more sensitive than conventional RT-PCR.In order to study the genetic resistance to AL on G1,G5,G6 and G8 lines, the chicks and embryos of G1, G5, G6 and G8 were inoculated with standard strain RAV-1 of ALV A subgroup and 6 chicks of G8 line and 9 embryos were randomly selected as control group. All died and remaining survival chicks at the end of experiment were necropsied to examine gross lesions. The body weight was tested from 4 weeks post infection and serum antibody titer of all chicks were tested by the ELISA detection kit of ALV. The virus copies per 107 lymphocytes (virus load) of chicks and liver cells of embryos were quantified by the duplex qRT-PCR assay. The resistance-related indexs of mortality, lesion scores, weight gain rate, antibody titer and virus load were used to assess the AL resistance of all infected lines. The results showed that the mortality of G5 was higher than other lines while G1 was the lowest. The body weight of G1 and G6 lines have increased more rapidly than G5 line. The antibody titer of G1 was higher than other lines from the 5 wpi to the end of the experiment while G5 was the lowest during this period. Furthermore, the antibody titer of G1 was statistically significantly (P<0.05) higher than G5 from 8 wpi to 11 wpi and from 15 wpi to the end. The lowest virus load was found on G1 from 7 wpi to 15 wpi and the virus load of G5 was statistically significantly (P<0.01) higher than G1 from 9 wpi to 13 wpi. G6 line has a neutral antibody titer among the infected lines. However,the virus load of G6 was statistically significantly (P<0.05) higher than G1 from 8 wpi to13 wpi and G6 maintained the highest virus load from 14 wpi to 18 wpi although no statistical difference was found among the infected lines. G8 line have a netural antibody titer and virus load compared with other lines. All these data indicated that the G1 line was able to produce a stronger genetic resistance to the infection of ALV while G5 line was more susceptible. In conclusion, this study not only provides a powerful diagnosis tool for the detection of ALV, but also provides significant evidence for studying the correlation between MHC and AL resistance. |
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