The Preparation Of The DNA Probe Of Chicken Inclusion Body Hepatitis Virus And Its Epidemiological Investigation

Avian inclusion body hepatitis (IBH) is one kind of acute infectious diseases caused by the avian adenovirus. The infected avian always died suddenly, autopsy shows severe anemia, jaundice, hemorrhage, necrosis and enlargement of liver, it can some times cause inclusion bodies within the liver cytoblast, The disease, also known as anemia syndrome. There are12serotypes related to the disease of avian adenovirus, there are10serotypes related to inclusion body hepatitis of natural outbreaks at least. Serotypes cited IBH are mainly type2,3,4,5,6,7and type8. the disease is found in Taiwan Province first in1976, after this, it is found Liaoning province, Hunan province, Jiangsu province, Jilin province, Henan and Inner Mongolia province have occurred, Showed a trend of increasing the spread, thus causing more and more attention. Dot blot hybridization becomes one kind of ideal method because of its simple operation and cost control, chick embryo is the best isolated cultures of chicken adenovirus. After inoculated by the yolk sac, embryos may die within2-7d, often seen in liver cells basophilic nuclear inclusion bodies,nucleus containing inclusion bodies are larged than normal, Can be reach to2to3times compared with the normal.Culturing standard strains of serotypes by chick embryo yolk sac inoculation, Design five pair of primer according to the conserve sequence of Hexon gene of Serotypes1,2,4,8,12and gene sequences of low homology between the5serotypes from NCBI GenBank, the proliferative virus through DNA extraction and PCR amplification, Then, through the retrieve of the target production from the gelatin, purification of the nucleic acid and fixed quantity, and then labeled it by digoxin kit, The labeled probe shows favorable sensitivity and specificity, The sensitivity assay indicates that the minimum content for detection is1pg and the specificity assay shows no cross reaction, and then detect some lesion liver tissue from some region of Shandong and jiangsu province by dot blot hybridization, All parts can detected positive disease chicken.To handle suspected illness material by of all the blood serums probe, and vaccinate7age SPF chicken embryos (0.2ml/only), to handle liver tissue of death embryo of inoculation48hours, and continued to transfering of culture for two generations in chicken embryos for purification virus, and then,retrieved DNA in allantoic fluid, detected it by the labeled probe, at the same time, taked Chicken embryos livers and fixed these by10%formalin for Histologic examination. Extracting DNA from allantoic fluid as templete, PCR for hexon fragments and sequencing analysis.According to the research, we can see the widespread of the pathogen of IBH. The popular serotype is mainly serotype2,4,8. the positive disease material by dot blot hybridization are Vaccinated chicken embryos, there are intranuclear inclusions visible through histopathological examination. The sequencing result of Hexon sequence compared with standard sequence of serumtype1,2,4,8,12respectively, sequence homology can reach to96percent above all, the highest can reaches98.8percent.