Establishment A Nd Application Of RT-PCR Detection Method For Subgroup JAvian Leukemia And Sequence Analysis Of Isolated Viruses

Avian leukemia(ALV-J)is an infectious disease widely existing in broiler and layer breeding industry,which mainly causes immunosuppression and neoplastic diseases.The subtype J avian leukemia(ALV-J)has the most widespread prevalence and the strongest pathogenicity.It is characterized by high virus-carrying rate,low morbidity and low mortality,which mostly cause growth and immunosuppression,resulting in lower production performance and great hanm to China’s aquaculture industry.At present,there is no effective drug or vaccine for avian leukosis.The population is purified mainly by detecting and eliminating the infected chickens,thus eliminating the spread of the disease from the root.Roosters can transmit virus to hens through insemination.ALV purification of roosters is the key to ALV purification in chicken farms.The most widely used traditional detection methods include ELISA,virus isolation,IFA immunofluorescence assay,etc.Each has its own advantages and disadvantages.This study established a RT-PCR(reverse transcription PCR)detection method,which is convenient to sample and fast to detect.It can effectively detect ALV in cock and hen.The positive detection rate is higher than the traditional detection method.This method has achieved good results through practical application.Study 1:Quantitative Analysis of Expression of Avian Leukosis Virus in Poultry TissuesIn order to understand the expression rules of ALV-J in various tissues and organs of poultry and determine the sampling materials for detecting ALV,three chickens with ALV-J disease were dissected,RNA of 14 tissues and organs such as hair follicle,heart,liver,spleen,lung,kidney,duodenum,jejunum and ileum,cecum,pancreas,cloacal epithelial tissue,fallopian tube,ovary,trachea and the like were extracted,reverse transcribed into cDNA,and then quantitative detection and analysis of ALV-J were carried out.The results showed that ALV-J was expressed in all tissues and organs of poultry,but the expression levels were different.Among them,cloacal epithelial tissues,hair follicles,fallopian tubes,ovaries,kidneys and other metabolic areas showed the highest expression levels.Therefore,cloacal cotton swab was selected as the detection material for ALV.Study 2:Establishment and Application of RT-PCR Detection MethodIn view of the highest expression of ALV-J in cloacal epithelial tissue,this experiment uses chicken cloacal cotton swab as the detection material for ALV-J,and establishes a RT-PCR detection method(reverse transcription PCR):RNA in sample diluent is extracted and transcribed back into cDNA.Finally,ALV-J specific primers H5 and H7 are used for PCR amplification and gel electrophoresis.Finally,the test results are judged.In this experiment,the annealing temperature and primer concentration were optimized for the established PCR method,and the optimal annealing temperature was 58oC,and the optimal primer concentration was 2.4μM.The RT-PCR method established in the experiment was used to detect laying hens in a large-scale chicken farm.The results showed that the positive detection rate of RT-PCR method was significantly higher than that of ELISA and virus isolation detection methods.Study 3:Detection of ALV-J Infection Rate in Roosters of Different BreedsNext,the RT-PCR method established by the experiment was used to detect ALV-J of 19 different breeds of cockerels with the same feeding conditions in a large-scale farm.1083 cockerels were tested,of which 258 were positive,with a total positive rate of 23.82%,while the positive rate of each breed was very different,ranging from 8.33%to 58.33%.Study 4:Sequence Analysis of Isolated VirusesIn order to understand the genetic information of epidemic strains in chicken farms and analyze the source and genetic variation of the strains,the ALV-J strains were isolated from the farms in Test 2 and Test 3,and sequence comparison analysis was carried out with domestic and foreign ALV-J reference strains to construct a genetic evolutionary tree.The results showed that the two isolated strains of ALV-J and all the reference strains of ALV-J belonged to the same big branch,and the subgroups A,B,C,D and endogenous subgroup E did not belong to the same branch,indicating that the isolated strains were subtype J ALV and not other subtypes.The other two isolates belong to a small branch with high similarity and close genetic relationship,while the two chicken farms are the relationship between introduction and seed supply,thus it can be seen that introduction is an important factor to spread the ALV-J.Study 5:Pathological necropsy of chickens infected with ALV and detection of MDV and REV coinfectionIn order to understand the clinicopathological changes caused by ALV-J infection and explore the pathogenicity and pathogenic characteristics of the isolated strains,8 chickens suspected to have died of ALV-J infection were subjected to pathological autopsy and their clinicopathological changes were observed.It was found that chickens infected with ALV-J were divided into obvious symptom type and no obvious symptom type.The obvious symptom types are chicken crown whitening,keel bending,Hepatomegaly,cardiac blood stasis,spleen necrosis,liver tumor,cecum enlargement,pancreatic tumor,mesenteric tumors,myogastric gland and stomach tumor,thymus swollen tonsils,etc.The asymptomatic type only shows Hepatomegaly,kidney enlargement,spleen swelling,etc.There are almost no other pathological changes and no tumor.