Isolation And Identification Of A Duck-Derived Fowl Adenovirus Serotype 4 And Its Pathogenicity To Goose

Hydropericardium hepatitis syndrome?HSS?is mainly a contagious disease of poultry caused by Fowl adenovirus serotype 4?FAdV-4?.The disease mainly affects 3 to 5 weeks old broilers,and its typical lesions are pericardial effusion and inclusion body hepatitis.Since June2015,the disease has been erupted in broiler flocks in many provinces of China,and has gradually spread from broiler flocks to laying hens,breeders and cherry-bred ducks,seriously jeopardizing the healthy development of China's poultry industry.In recent years,there have been reports of goose HHS infection in a few gooses in China,but no in-depth research has been conducted.In this study,we isolated a FAdV-4 from a naturally infected diseased duck and designated it as SDTA.We determined its pathogenicity to goose by animal experiments,confirming that duck avian adenovirus type 4 can cross-infect goose.It shows a certain pathogenicity and provides certain epidemiological data for the prevention and control of the disease in poultry production.The main research contents are as follows:1.In this study,chicken embryo yolk sac inoculation method was used to isolate the virus from the liver tissue of commercial ducks suspected of FAdV-4 infection in Taian,Shandong Province.The isolated strains were tested by PCR amplification,hemagglutination test,EID₅₀test,purity test and Hexon gene sequencing and analysis.The results of PCR showed that the strain was group I fowl adenovirus serotype 4;the purity test showed that the strain could not agglutinate chicken red cells,and the common virus was negative by PCR,indicating that the purity of the strain was better;the EID₅₀ of the virus was 10^-7.15/0.2 mL;the Hexon gene of the isolate was sequenced,and the sequence was compared with the published group I fowl adenovirus serotype 4 gene for nucleotide homology and genetic evolution analysis,indicating that the isolate was found to be 99.9%homologous to the two isolates of the Chinese mainland,and the homology with the strains of South Korea,Europe and America was 98.3%to 98.6%,which was relatively far.2.In order to investigate the pathogenicity of FAdV-4 isolates to geese,225 1-day-old Taizhou geese?75 as control geese and 150 as infected geese?were randomly divided into nine different groups?6 as experimental group and 3 as negative control group?.At the age of 10,20and 30 days,geese in six experimental groups were artificially infected by oral and subcutaneous injection respectively.Each geese was inoculated with 1 mL allantoic fluid containing virus.The geese in the control group were inoculated with phosphate buffered saline?PBS?.The geese were observed for 15 days,and the clinical symptoms were recorded.At 3,6,9,12 and 15 dpi,three geese in each group were randomly killed and their tissues,organs and blood were collected.Some tissues and organs were stained with HE to observe the histopathological changes.The other part was extracted with DNA to detect the viral load in each tissue by fluorescence quantitative PCR.Blood was collected and serum was collected to detect blood biochemical indicators?ALT,AST,LDH,ALP and UREA?and cytokines?IL-2and IFN-??.Geese in the 10-day-old subcutaneous challenge group showed obvious clinical symptoms,including depression and paralysis.The symptoms of necropsy showed pericardial effusion,liver swelling,hemorrhage and yellowing,thymic hemorrhage and swelling,lung edema and hemorrhage.Kidney hemorrhage and swelling,FAdV-4 was less pathogenic to the goose in the oral challenge group and the 30-day-old subcutaneous challenge group,and did not show obvious clinical symptoms and necropsy changes.Histopathological findings showed myocardial interstitial edema and mild inflammatory cell infiltration;basophilic inclusion bodies appeared in the nucleus of the liver;lymphocytes decreased and focal necrosis in the spleen;pulmonary artery wall thickening and pulmonary capillary congestion;The kidneys have a large number of vacuolar necrosis,glomerular swelling and hemorrhage,tubular detachment and inflammatory cell infiltration;both bursa and thymus show lymphopenia and interstitial edema.Real-time PCR results showed that the virus was detected in all organs collected.The virus content in liver and kidney was higher.The viral load of small-day-old geese was higher than that of large-day-old geese.The results of cytokine levels showed that the levels of IL-2 and IFN-?first increased and then decreased,indicating that geese had a strong immune response to FAdV-4 invasion.The results of blood biochemical indicators showed that the indicators of the 10-day-old challenge group were significantly higher than the control group,indicating that FAdV-4 caused serious damage to the heart,liver and kidney of the geese.The indexes of 20-day-old challenge group were slightly lower than those of 10-day-old challenge group,but there was no significant difference between 30-day-old challenge group and the control group.The above results indicate that goose has an age-related resistance to FAdV-4 infection.