| Avian leukosis(AL), which is caused by avian type C retrovirus group, is the general designation of many kinds of avian tumor diseases. In the poultry industry, AL has lead to benign and malignant tumors, subclinical, growth retardation, egg drop and immunosuppressive. In recent years, avian leukosis has widely disseminated in the flocks in China, which has been a serious threat to the safety of local varieties of chicken and brought great difficulties to the conservation and breeding work in local varieties of chicken.Currently, the international classical method of cleaning AL results in missed detection due to a single detector material or detection methods. In this study, virus of sterile anticoagulant serum from cocks and hens in local varieties Luhua chickens were isolated by cell culture.ALV-p27 antigen in supernatants and egg proteins were tested by ELISA, in order to understand the infection status of ALV in Luhua flock. And randomly selected 5 copies CEF which are ALV-p27 antigen positive to extract cDNA, then the ALV-J primer pair was used to identify virus by PCR.At the same time, detect the different materials of the same batch of chicken with different methods, which aims to explore the relevance of different materials with different detection methods and optimize ALV detection methods for reducing ALV missing rate.Tracking tested the offsprings of the breeder flocks in order to achieve the decontamination work of AL in Luhua. In addition, the correlation of the ALV infection status of the chickens and NDV, H5, H9 antibody levels of the chickens and the chickens’ positive rate of Pullorum was researched in this paper.1 Investigation of Luhua flock infection statusTo acquaintance the ALV infection status of the chickens in a farm in Jiangsu, 1024 hatching eggs from the farm flocks(two eggs of each 508 chicken, one egg of each eight chicken) and the corresponding 516 hens sterile anticoagulant serum, 232 supernatant copies cock sterile anticoagulant serum and semen were inoculated to 9d CEF, ELISA was performed to detect ALV-p27 antigen. The results showed that hens protein positive rate was20.7%(107/516), hen sterile anticoagulant serum positive rate was 2.52%(13/516), and the protein-positive samples can not completely covered the positive a sterile anti-hen clotting positive samples.The positive rate of semen was 15.1%(35/232) and sterile anticoagulant was 1.29%(3/232). Exogenous ALV-J(LH14J) was identified in the same chicken by PCR.the test results showed that there is a certain degree of Luhua chickens infected with exogenous ALV. The hens’ protein and the sterile anticoagulation, these two materials can’treplace each other.2 Research of ALV detection materials and methods OptimizationIn order to avoid the missed detection phenomenon of using single materials and methods, isolating RNA from hens’ anticoagulation sterile serum for dot blot hybridization experiments. In addition, 232 copies of correspondence were inoculated with CEF cell culture.9days later, testing supernatant with the ALV-p27 antigen ELISA test.The results showed a positive rate of rocks and hens dot blotting is 5.88%(44/748), in which the coincidence rate between hen blood dot blotting and protein was 30.0%(6/20), and the coincidence rate with hen blood CEF supernatants positive was 30.0%(6/20). The coincidence rate of rooster blood spot hybridization and the blood CEF supernatants was 12.5%(3/24). The positive rate of rooster semen was 15.1%(35/232),and the coincidence rate with the blood of its CEF supernatants The positive rate was 8.6%.The results of the study showed that, the single way of testing protein, anticoagulation or semen could not detect all ALV positive chickens. The complement of different detection materials with each other were used to ensure the positive ALV detection rate of chickens. In the process of purifying ALV, in addition to the classical ELISA detection of the virus from cell culture of Luhua hens’ protein and chickens’ anticoagulant, it is a powerful complement of the classical RNA purify method by adding measuring semen, RNA isolation from serum and dot blot hybridization Moreover, there were three kinds of modalities in the 107 positive chickens, which were tested by protein. The number of the chickens with two eggs of each is 93, covering 86.9%. The number of the chickens with one negative egg and one positive egg is 12, covering 11.2%. There were two chickens with only one positive egg, covering 1.9%. This study showed that increasing the number of the samples during the ALV testing will induce the miss rate of ALV.3 Track detection of the offsprings of breeder flockIn order to understand AL purification effect of the chicken group, and this study collected 1320 copies of sterile anticoagulant serum from offspring of the chicken group for virus isolation,the results show that the positive rate of the parental chickens was 0.3%(4/1320), the positive rate was lower significantly compared to their parent flocks which the positive rate was 2.14%(16/748).The above data indicating that the purification work of AL in Luhua has obtained certain achievement.4 Research of the relativity between Luhua flock infection status and vaccines of NDV, H5, H9 antibody levels of the chickens and the chickens’ positive rate of PullorumTaking 15 eggs of both negative and positive ALV-p27 antigen from different gradients OD value(negative, OD0.2-0.3, OD0.5-0.6, OD0.8-0.9, OD1-2, OD2 above), and testing NDV 、 H5 、 H9 antibody titers of samples from each group. The results showed that in Luhua flock NDV, H5, H9 antibody levels of the p27 antigen positive were separately lower than those of the negative, and the difference was significant, the other antibody levels were not significantly correlate.But the overall NDV antibody levels of positive eggs were lower than those of negative eggs(8.9±1.106 VS 9.8±0.145), the difference was significant. There was no significant difference of H5 antibody levels between ALV positive and negative eggs(7.9±0.457 VS 8.2±0.223).The H9 antibody level of positive eggs was slightly lower than those of negative eggs(9.8±0.674 VS 10.5±0.192), the difference was not significant, but there were significant differences in the trend(p = 0.063). In addition, detecting salmonellosis infection of eggs in each group. The results showed that Pullorum positive rate of p27antigen-positive chickens was 39.6%,the negative chickens’ was 22.2%,the pullorum disease detection rate of p27 antigen-positive chickens was higher compared with negative chickens,but there was no significant difference of the pullorum disease detection rate in different values of the gradient OD p27 antigen positive chickens, with no significant correlation. |
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