Isolation And Identification Of Novel Goose Astrovirus And Its Pathogenicity To Chickens And Ducks

In recent years,avian astrovirus has been increasingly reported and its infection in poultry such as chickens,ducks and geese has become very common.The outbreak and quick spread of the novel goose astrovirus(N-GAst V),primarily causing gosling gout,caused huge economic losses to the Chinese goose industry since 2016.Thus far,increasing reported evidences have indicated that the potential cross-species transmission of N-GAst V between domestic fowl is possible.The N-GAst V has the potential to adapt to new hosts,and infected new hosts exhibit symptoms similar to those of infected goslings.In the study,9 strains of N-GAst V were isolated from clinically suspected diseased samples.The isolates were identified and analyzed to confirm the variation of the N-GAst V.The dominant strains were selected for pathogenicity studies on SPF chickens and meat ducks.1.Isolation and identification of N-GAst VPCR testing was performed on some of the suspected infected samples collected from2021 to 2022 in our laboratory,and the positive samples were isolated using chicken embryonic liver cells(CEL).9 N-GAst V were isolated.Among them,3 strains were of duck origin and 6 strains were of goose origin.The results of sequencing analysis showed that the nucleotide homology of the 9 isolates ranged from 97.0%to 99.8%,and the nucleotide homology with the classical strain SDPY ranged from 97.5%to 99.1%,and were on the same branch of the genetic evolutionary tree,indicating that the isolates did not undergo significant mutations.The isolates are in different branches from the classical turkey,duck and chicken origin astroviruses and are more distantly related.The strain SDTZ2021 was selected for animal pathogenicity study and animal regression test.After 0.3 m L(TCID₅₀=10^-5.14/0.1 m L)of viral solution was administered intravenously to 1-day-old fledgling geese,the goslings showed clinical symptoms such as depression,lying down and moving around,and reduced feeding.Through autopsy,it was found that there were pathological changes such as urate adherence and deposition on the heart,liver surface and inner thigh muscles of the dead geese,kidney enlargement and bleeding,ureter and glandular stomach have urate deposition.2.Pathogenicity of N-GAst V in SPF chickensA total of 160 1-day-old healthy SPF chickens were randomly divided into four groups:oral infection group,subcutaneous infection group,intramuscular infection group and control group.Each group has 40 chickens.Infected group was inoculated with 0.6 m L(TCID₅₀=10^-5.14/0.1 m L)of viral fluid at 1 day old,and the control group was inoculated with 0.6 m L of sterile saline,and the clinical symptoms and autopsy lesions of SPF chickens in each group were observed.At 1 d,3 d,5 d,7 d,9 d,11 d,14 d,17 d,20 d,24 d,28 d,33 d,38 d and 44 d after infection,weighing three randomly selected SPF chickens to monitor the change of body weight.Three cloacal swabs,three non anticoagulated and anticoagulated blood samples were collected to monitor the the rule of detoxification,viraemia and serum antibody level changes of SPF chickens.The heart,liver,spleen,lung,kidney,duodenum,thymus,glandular stomach,cerebrum,bursa and pancreas were collected for the preparation of pathological tissue sections,virus distribution and load detection.The results showed that the infected SPF chickens showed signs of depression,bunching,drowsiness and white stools at 4 dpi;the liver and kidneys were enlarged and bleeding,the thymus gland was bleeding,and a small amount of urate was deposited on the surface of the kidneys and in the ureter.The infected group was all lower in body weight than the control group and grew slowly,with subcutaneous infection group being the most significant.The Histopathological results showed that the infected SPF chickens showed myocardial hemorrhage,myocardial fiber rupture and widening of gaps;hepatocyte necrosis with a large a large number of inflammatory cell infiltration;lymphocyte disintegration,necrosis,splenomeloblast destruction and a large number of red blood cells distribution;renal interstitial capillary hyperemia,renal interstitial structure destruction,accompanied by inflammatory cell infiltration;and a large number of red blood cells appeared in the thymus cortex and medulla.The results of viral tissue distribution and load detection showed that the N-GAst V has a wide range of tissue addiction,which can be distributed in all tissues and organs of SPF chickens,and the organs with the highest viral content in each infection group are livers.Viremia and detoxification rules in all infection groups were basically the same,and detoxification occurred in the early stage of infection,reaching a peak of 7 dpi in the oral infection group and cervical subcutaneous infection group,and reaching a peak of 5 dpi in the intramuscular infection group,and then showing a downward trend.Combined with the viral load of tissues and organs,viremia and detoxification,after artificial infection with SDTZ2021 strain of virus,the viral content in SPF chickens showed a trend of first increasing and then decreasing with the increase of infection age,reaching the highest at 5 to 7 dpi.The antibody levels of each infection group showed a trend from high to low,and the difference between them and the control group was highly significant(P<0.01).3.Pathogenicity of N-GAst V in meat ducksIn this study,the experimental design was based on different infection age and infection mode:the 1-day-old infection group was labeled A,the 7-day-old infection group was labeled B,and the 14-day-old infection group was labeled C;the oral infection group was labeled 1,the cervical subcutaneous infection group was labeled 2,and the leg intramuscular infection group was labeled 3.400 healthy 1 day old were randomly divided into 10 groups:A1,A2,A3,B1,B2,B3,C1,C2,and control groups,with 40 per group.Each duckling in the infected group was inoculated with 0.6 m L of SDTZ2021 virus fluid(TCID₅₀=10^-5.14/0.1 m L),and each duckling in the control group was inoculated with 0.6 m L of sterile saline,and the clinical and autopsy symptoms of each group were observed.The monitoring of body weight and sample collection were the same as the pathogenicity test for SPF chickens.The results showed that all infected groups had symptoms of loss of appetite and white stool at 1 dpi.Death occurred in the infected group from 2 to 4 dpi,and the peak of death reached in 7 to 12 dpi,with a mortality rate of 5%to 20%.The 7-day-old infection group(group B)and the intramuscular infection group(group 3)had the highest number of deaths,while the 7-day-old intramuscular infection group(group B3)had the highest mortality rate.The changes in autopsy were basically the same in all infected groups,showing hepatic and renal enlargement and hemorrhage;urate deposition in the ureter;splenomegaly enlargement was mottled;among them,the 7-day-old intramuscular infection group(group B3)had the most serious lesions,and white urate deposition appeared on the surface of internal organs such as the heart.The weight of the infected groups was lower than that of the control group and the growth was slow,with the most significant in the intramuscular infected group(group3).The histologic changes of pathology in all infection groups were basically the same,which were manifested as:myocardial fibroedema,calcium salt deposition around myocardial fibers,swelling and necrosis of hepatocytes,hepatocellular cord disorder,lymphocyte disintegration and necrosis in the spleen tissue,filled with a large number of red blood cells;glomerular capillary atrophy,blue stained calcium salt mass particles visible in some capillaries,severe hyperemia of the interstitial,atrophy of the renal tubules,and severe congestion of the interstitium.The detection results of viral tissue distribution and load showed that the N-GAst V could be distributed in all tissues and organs of meat ducks,and the organs with the highest viral content in each infection group were kidneys.The 7-day-old intramuscular infection group(group B3)ducklings had the highest viral content of all tissues and organs.The changes in viremia and detoxification patterns were basically the same in all infectious groups,and detoxification occurred in the early stage of infection,and the highest peak occurred in the intramuscular infection group(group 3).The antibody levels of each infection group showed a trend from low to high,and the difference between them and the control group was highly significant(P<0.01).In summary,after artificial infection with SDTZ2021strain,the virus level in the infected ducklings showed a trend of increasing,then decreasing and then increasing with the increase of age,except for the ducklings in the 1-day-old oral infection group(group A1),which reached the peak at 7 dpi.The second peak load was generally lower than the first peak load.