Isolation,Identification And Vertical Transmission Characteristics Of Group Ⅰ Fowl Adenovirus In Ducks

Group I Fowl Adenovirus（FAdV-I）,belonging to the genus Avian adenovirus of the adenoviridae family,is a double-stranded DNA virus with non-envelope.FAdV is a typical icosahedral symmetrical structure,which can be divided to 5 genuses and 12 serotypes:A genus（FAdV-1）,B genus（FAdV-5）,C genuses（FAdV-4,FAdV-10）,D genuses（FAdV-2,FAdV-3,FAdV-9,FAdV-11）and E genuses（FAdV-6,FAdV-7,FAdV-8a,FAdV-8b）.FAdV-I mainly causes hydropericardium-hepatitis syndrome（HHS）,inclusion body hepatitis（IBH）,Gizzard Erosions（GE）and other diseases.Firstly Chinat had an outbreak of HHS in chickens,various serotypes FAdV-I are also widely spread in waterfowls,which seriously restricts the development of poultry industry.FAdV-I can transmit horizontally,the main medium are air and excreta.But we found that ducks infected with FAdV-I,egg production declined,the number of dead embryo increased during hatching,ducklings broke shells difficulty and retarded growth.It is speculated that FAdV-I can be infected vertically through duck-egg-embryo.Virus proliferation during hatching leads to embryonic death or infecting virus.Ducklings out of the shell developed slowly,because they infected virus during hatching.In order to confirm whether FAdV-I can transmit vertically in ducks,this experiment carried out a study on the vertical transmission characteristics of breeding ducks to provide an epidemiological basis for disease prevention and control.1.Detection and isolation of FAdV in eggs,duck embryos and ducklingsIn this study,FAdV-positive cases in breeding ducks were detected from seven hatcheries in Shandong,Inner Mongolia,Jiangsu,Hebei and so on during 2022-2023.A total of 600samples were collected from fresh eggs,dead embryos and weak ducklings,the vitelline membranes were collected aseptically from the eggs,the liver and spleen tissues of the duck embryos were collected for FAdV detection,and the positive rate results were as follows:Taian4%（12/300）;Jiyang 10%（2/20）;Jiangsu 16%（8/50）;Ningyang 11%（11/100）;Cangzhou 12.5%（5/40）;Liaocheng 10%（3/30）;Inner Mongolia 11.7%（7/60）,the overall positive rate was 8%（48/600）.The positive samples were made into tissue homogenate,after virus supernatant centrifugation,10 strains of FAdV were isolated and identified by inoculation of 8-day-old SPF chicken embryos.Through the homologous comparison and genetic evolution analysis of the isolate Hexon gene,4 strains were serotype 4,6 strains were serotype 8b.2.Vertical transmission characteristics of FAdVAfter the 250-day-old Pekin Duck were detected negative for FAdV antibody and detoxification,they were divided into five groups A-E according to the appropriate proportion,each group was 16 ducks and 4 drakes.The ducks were fed in isolation until the breeding ducks adapted their new environment（egg production rate reaches 30%）.Group A and D were intramuscular injection groups,Group B was oral group,FAdV-I was represented by the FAdV-8b strain(TCID₅₀=10^-5.77/0.1 m L),each group was inoculated with 3 m L.Group C and E were control groups,injected with 3 m L normal saline.Collecting eggs and hatching to study ducklings infection in groups A,B and C.Studying FAdV infection of breeding ducks in groups D and E.After the breeding ducks were inoculated with FAdV,three breeding duck were randomly selected for collecting serum and cloacal every third day,the level of FAdV antibodies in the serum was detected by ELISA method,the IFN-γand IL-6 levels in the serum were monitored by cytokine detection kit,and the detoxification of breeding ducks was detected by PCR.Two ducks were dissected every forth day,the main organs such as heart,liver,spleen,lung,kidney,glandular stomach,follicles and fallopian tubes were collected aseptically for pathological research.After the breeding duck infected virus,they were depressed and occasionally excreted thin white feces,the number of eggs decreased,the number of Sand-shelled eggs,soft-shell eggs and bloodstained eggs increased.The autopsy results showed that:Heart hemorrhage,liver hemorrhage,hepatomegaly,yellowing,fragility,tubal hemorrhage,severe ovarian atrophy,kidney hemorrhage,hypernephrotrophy.The results of FAdV antibody level detection showed that after 1 dpi,the antibody level of the infected group gradually increased,the level gradually decreased after the peak of 10 dpi.Antibody levels in the control group were negative.The cytokine detection results showed that the IFN-γlevel of the infected group gradually increased,the level decreased after the peak of 7 dpi and stabilized at 12 dpi,IL-6 level gradually increased and decreased after the peak of 10 dpi.The level of IFN-γand IL-6 in the control group tended to be stable.The results of histopathological tests showed that liver cells had inclusion bodies,degeneration,cardiac interstitial edema,ovarian lymphocyte infiltration,etc.The results of the detoxification detection showed that detoxification occurred at 4 dpi in the intramuscular injection group and 6 dpi in the oral group,there was no detoxification in the control group until the end of the experiment.The above test results show that after FAdV infected breeding ducks,the body was stimulated to produce an immune response,the impact on the liver and ovaries was more serious,which impaired the egg production ability of breeding ducks.The ducks had a long detoxification time through the cloaca,which creates favorable conditions for the spread of the virus on the eggs.The eggs were hatched in different groups,hatched ducklings were raised,regularly dissected and collected tissue and serum,the hatching results showed that groups A,B and C produced 521 eggs during the experimental（intramuscular injection group/oral group/control group=148/162/211）,the egg number of infected group decreased at the begining of the experiment and returned to normal after 10 d.The eggs which were not suitable for hatching in each group were removed,100 eggs were randomly selected for hatching in every group,the results showed that the fertilization rate of the intramuscular injection group was 76%（76/100）and the hatchability rate was 65.8%（50/76）.The fertilization rate of the oral group was 87%（87/100）and the hatchability rate was 70.1%（61/87）;The fertilization rate of the control group was 90%（90/100）and the hatchability was 88.9%（80/90）.The positive detection rate of viruses in eggs,duck embryos and ducklings showed that 68.8%（66/96）in the intramuscular injection group;41.1%（44/107）in the oral group;FAdV was not detected in the control group.The results of pathological changes of hatching ducklings showed that the dead embryos showed fuzzy shape,yolk dispersion,and embryo form blood film in the early stage（1-5 d）.In the middle stage（5-15 d）,the embryo body was dysplasia,hyperemia,edema,yolk malabsorption and liver hemorrhage;In the later stage（after 21 d）,it is mostly difficult for ducklings to come out of their shells.The test results of FAdV antibody level in hatching ducklings showed that the antibody level was highest at 1d,then the level gradually decreased and tended to be stable after 10 days.Cytokine detection results of hatching ducklings:IFN-γand IL-6 levels were highest at birth,the level gradually decreased and stabilized after 10 dpi.FAdV was isolated from the positive sample tissue,the isolated strain Hexon gene was sequenced,the homology with the seed strain was 99.1%-100%.The above results show that FAdV-I can be transmitted from infected breeding ducks to offspring through eggs,FAdV-I can be transmitted vertically on breeding ducks.