Isolation And Identification Of Fowl Adenovirus Species C K Strain And Establishment Of TaqMan Probe Real-time Quantitative PCR Assay For Detection

Fowl adenoviruses can be subdivided into three groups: group I,group ?,group ?.Then fowl adenoviruses can be subdivided into five species(FAd V-A to FAd V-E),basing on molecular structures,and 12 serotypes(FAd V-1 to FAd V 8a and 8b to 11),basing on cross-neutralization test results,which are all over the world.All the FAd V 12 serotypes can induce inclusion body hepatitis(IBH)syndrome and mortality is approaching 10%,which is characterized by hepatic necrosis.FAd V serotype 1(FAd V-A,chicken embryo lethal orphan virus)can also induce gizzard erosion(GE).Inclusion body hepatitis-pericardial effusion syndrome(HHS)is the most serious disease caused by FAd V,most of which is caused by fowl adenovirus serotype 4 and 8.In 1987,hydropericardium syndrome firstly appeared in Pakistan,then spread around the world.Since 2013,there had been cases of inclusion body hepatitis and hydropericardium syndrome in domestic farms.The number of Chinese provinces infected by FAd V was increase in 2015,with sudden onset among flocks and a high mortality of 30%~70%,which caused serious economic loss to the domestic chicken industry.In recent years,the number of HHS cases in various parts of the world has also been on the rise,such as India,Canada,Japan,Korea.In 2015,outbreak of HHS occurred in a chicken farm in Henan Province.The liver and pericardial effusion of the infected chicken were collected in this study to reserach its pathogenic mechanism.Supernatant fluid was extracted after grinding,then was separated and purified by inoculating CEL,could see cytopathogenic effect and the shape of grape string with dropping out.The isolated strain was named K strain.The purified viral DNA was extracted,PCR amplification was performed,the amplified product was sequenced.The amino acid sequence homology analysis and genetic evolution analysis between K strain and other adenovirus in Genbank showed that the homology of K strain were isolated in this study and C species and serotype 4 of fowl adenoviruses was 93.6%,the relationship was closest,so K strain was FAd V species C serotype 4.In this study,K strain was inoculated to 1 day-old,64 day-old,220 day-old SPF chicken,and the all mortality was 100%,which indicated that K strain was highly pathogenic.Oil-emulsion vaccine preparated by 20% liver tissue homogenate inactivated by formalin and ISA71 VG adjuvant high-speed shearing,in order to explore the virus minimum amount of vaccine effectively preventing HHS,oil emulsion vaccine,106TCID50,105TCID50,104TCID50,103TCID50 respectively,immunizing 3 week-old SPF chicken,and setting up the control group.After 4 weeks immunized,each group was inoculated with the K strain of 106 TCID50,observing the mental state of the chicken daily,recording the time of death until 14 days post-infection.According to attacking protection test results,showed that the preparation of group A is minimum amount,the vaccine containing 106 TCID50 virus could achieve complete protection for flocks.This showed K strain had good immunogenicity.K strain could be used as a strain of candidate for preparing vaccine to prevent fowl adenovirus infection.In order to be suitable for FAd V clinical mass detection,preparativing standard plasmid basing on K strain was used to establish the method of TaqMan probe fluorescence quantitative polymerase chain reaction(PCR)to detect FAd V-4.Amplification efficiency of the method was as high as 93.87%,the correlation coefficient was 0.998 closely to 1.It was high specificity and good repeatability,minimum detection of FAd V template concentration was 2x101 copies/u L,it was 10 times more sensitive than the conventional PCR.It was great significance to promote the healthy development of poultry industry.At the same time,the biological characteristics of K strain were studied.Applying this fluorescence quantitative polymerase chain reaction method detected tropism of K strain to different organizations of 3 week-old SPF chicken infected,the results showed that 3 week-old SPF chicken infected by K strain,the virus widely distributed in organization of the chicken,the virus copy numbers can be detected in 3dpi,virus copy numbers was the highest in 8 dpi,the virus copy numbers of liver was up to 2.208x108 copies/g,the virus copy numbers of heart was 2.723×106copies /g,and the least amount of virus in the thymus was 8.710 x104copies/g.So K strain is extensive tropism in the majority of tissues in infected chickens and liver were the main troposim tissue.