Pathogenicity Analysis Of An H9N2 Subtype Avian Influenza Virus Isolated From Chickens In Guizhou Province To Chickens,Ducks And Mice

Low pathogenic avian influenza?LPAI?is prevalent among birds,and this virus has been isolated from a variety of mammals.Chicken,duck and mouse were used to study the cross-breed animals infected with low pathogenic avian influenza in order to comprehend the transmission of different breeds of livestock infected by low pathogenic avian influenza,and understand the mechanism of low pathogenic avian influenza.METHODS:24 52-day-aged black silky fowl chicken,24 52-day-aged cherry valley duck and 24 6 to 8-week-aged Kunming mice were divided into four groups.Six animals in each group were randomly selected and classified according to 0day,3 days,6 days and 9 days.These animals were injected with 0.2 mL PBS on 0 day,then animals were injected with 10^7.0 EID₅₀/0.2 mLH9N2 subtype avian influenza virus.Visceral tissues and blood samples were collected on 0 day,3 days,6 days and 9 days.?1?The clinical and anatomical pathological symptoms were observed,the hem agglutination titers of the three animals were measured by hemagglutination test,the lung coefficient was measured,the blood physiological indexes of the three animals were measured by three kinds of cell analyzer,and the IgG antibody level was detected by immunoglobulin G?IgG?ELISA kit.?2?The pathological changes of lung and trachea were observed by hematoxylin-eosin?HE?staining.?3?Virus content in lung tissues of three animals were tested by Quantitative Real-time Fluorescence PCR?RT-PCR?.?4?Mast cells in lung,trachea and other tissues were observed by toluidine blue?TB?staining.?5?Tumor necrosis factor?TNF-a?,interleukin-2?IL-2?and histamine?HIS?in blood was detected by ELISA kit.?6?The transcription of calmodulin?CaM?in lung tissue was detected by RT-PCR,and the protein content of CaM in lung tissue was detected by ELISA kit.Results:?1?The hemagglutination titers of the infected group were more than 2⁴and those of the control group were less than 2⁴.The RT-PCR of infected animals had amplification curves,but the blank control had no amplification curves.?2?The white cells of chickens was decreased significantly on 9 days?P<0.05?,that of ducks was increased significantly on 3 days?P<0.01?,and that of mice was increased significantly on 6 days and 9 days?P<0.01?.Red blood cells of chicken was decreased significantly on 9 days?P<0.05?,that of ducks was increased significantly on 3 days and 6 days?P<0.01?,that was increased significantly on 9 days?P<0.05?,that of mice was increased significantly on 3 days?P<0.05?.Platelets of chicken were increased significantly on 6 days?P<0.05?,that of ducks was increased significantly on 3 days and 6 days?P<0.01?.?3?After infection,chickens were showed strong spirit and loss of appetite.Enlargement of thymus and lung,pleural effusion and obvious red blood cells was seen in alveolar cavity,a large number of neutrophils was found in lung tissue,red blood cells and exudates was seen in trachea.Ducks was found with pleural effusion,pulmonary enlargement.Bleeding and a large number of neutrophils was found in lung tissue of ducks.Mice were found depression,loss of appetite,and lung swelling and splenomegaly.Red blood cells were seen in lung tissue of mice.Chickens,ducks and mice had the highest pathological score on the 6th day.The pathological scores of chickens,ducks and mice increased significantly at 3 and 6 days after infection?P<0.01?.At 6 days,the pathological scores of ducks and mice were the highest.At 9 days,the pathological scores of ducks and mice decreased significantly?P<0.05?.At 6 days,the pathological scores of chickens were significantly higher than those of ducks?P<0.01?,but significantly higher than those of mice?P<0.05?;?4?Mast cells in lung,trachea,intestine,bursa of Fabricius and thymus tissues of chickens and ducks were increased significantly after infection.The number of MC in trachea,intestine,bursa of Fabricius and thymus tissues increased significantly?P<0.01?,and the number of MC in spleen tissues increased significantly?P<0.05?;?5?Lung tissues of three animals had the highest toxicity on the third day,and the concentrations of virusin chickens lungs were most in the three animals.?6?IL-2 and TNF-?in the blood of chickens,ducks and mice had the highest concentration after infection.?7?The gene level of CaM transcription in lung tissues of three animals was increased significantly?P<0.05?,but the protein expression of CaM in lung tissues was decreased significantly?P<0.01?.Conclusion:1.It is confirmed that low pathogenic avian influenza can be transmitted across breeds of livestock,chickens are susceptible,ducks are next,and mice are least susceptible.2.Low pathogenic avian influenza can cause mainly pathological damage to animal lung tissue,and the degree of pathological damage is related to susceptibility and animal species.3.The inflammatory reaction was induced by low pathogenic avian influenza with inflammatory factors.4.Low pathogenic avian influenza can up-regulate the gene level of CaM transcription in lung tissue,but may inhibit its protein expression in lung tissue.