| Avian influenza virus(AIV), a kind of single-strand negative virus, belongs to Orthomyxoviridae family. AIV can infect a variety of poultries and mammals which includ humans. Since the first isolation from chicken was reported in Guangdong province in 1994,H9N2 AIV has been very popular in China and became the main subtype of low pathogenic AIV(LPAIV). Though the pathogenic of H9N2 AIV is lower, poultry infected with H9N2 AIV usually showed respiratory symptom and heavy egg production drop in egg-laying hens. The morbidity and mortality rates usually became higher when the infection accompanied by bacterial or other viral infection, leading to serious economic loss in poultry farming.Although H9N2 subtype is a low pathogenic avian influenza virus, it has caused huge economic losses in our country. Waterfowl, especially ducks and geese are considered to be the principal natural reservoir for AIV and play a critical role in the transmission and evolution of AIV. In all 8 segments of AIV genome, the HA gene has important functions in viral pathogenicity, antigenicity and host specificity of the virus, and it is the most volatile one.Therefore, it is of great importance to carry out phylogenetic analyses of the HA genes mutation of H9N2 subtype AIV and investigate the pathogenicity of a H9N2 AIV for geese.1. Isolation and identification of H9N2 AIV and phylogenetic analysis of HA geneSuspected avian infetion(AI) were observed in chicken farms in Shandong province in China during 2012 to 2013. Lungs samples of infected chickens were collected and disposed normally, and inoculated into allantoic cavity of 9 days-old SPF chick embryos. Finally, 25 strains of H9N2 AIV were isolated and identified. Total HA gene of 25 strains H9 AIV were amplified by RT-PCR and sequenced for phylogenetic analysis. Phylogenetic tree was drew by MEGA software(version 5.10). As a result,(1) HA gene of 25 strains H9 AIV shared94.5%-100.0% nucleotide homology and 93.8%-100.0% amino acids homology.(2) All of the25 strains H9 AIV belonged to sub-branch Y280-like of Eurasian sub-branch.(3) HA protein of 25 strains H9 AIV had seven to nine potential glycosylation sites. The deletion potential glycosylation sites in position 218 and an additional potential glycosylation sites in position145 in HA protein were also found.(4) All the cleavage site of HA protein was RSSR↓GIF,which was the characteristic of low pathogenic AIV strain.(5) Receptor binding site of 25 H9AIV strains were conservative. The 234 aa in HA gene was L(Leu),which is the characteristic of the ability to binding with α-2,6 receptor in human beings and special focus should be paid in public health as a result of the popular of these strains.2. Pathogenicity of H9N2 subtype AIV for geeseA goose-original H9N2 AIV(gAIV-H9N2) which was isolated, identified and saved by our lab was used for experimental infetion. 60 2-week-old geese were randomly invided into intravenous injection group, intranasal infetion group and control group. The experimental groups were inoculated with 1 m L g AIV-H9N2 allantoic fluid(ELD50 10-7.36/0.2m L) in intravenous injection and intranasal drip, respectively. The control geese were inoculated with the same volume of sterile physiological saline. The control and experimental groups were fed separately and clinical manifestation was observed every day after infection. At 3, 6, 9, 12, 15,21 and 28 day post inoculation, blood of geese were collected for the serum to measure the changeable rule of H9 antibodies by HA-HI. Cell factor IL-2, IL-6, IFN-β, IFN-γ were detected by ELISA detection kit and blood biochemical index were detected at the same time.At 3, 6, 9, 12, 15 day post inoculation, 3 geese in each group were chose randomly and killed for the detection of pathological anatomical change. Brains, livers, lungs, pancreas, spleens and other tissues were collected and fixed into 4% formalin solution to be made into section and dyed.As a result,(1) The clinical symptom of geese in experimental groups were depression,drop in feed and water intake, white loose stools and slow-growing and some respiratory symptoms. Obviously, geese in intravenous injection group presented more sevious clinical signs than geese in intranasal infection group. There were no geese died during the infection.All geese got right from 9 day post inoculation on. The control groups had no obvious symptoms.(2) Hemorrhage in lungs and epicardium were found at 3 day post inoculation in intravenous injection group and intranasal infection group. Hemorrhage in lungs and glandular stomach were found at 6 day post inoculation in intravenous injection group and intranasal infection group. Hemorrhage in lungs and livers, swell and necrosis in spleen were found at 9 day post inoculation in intravenous injection group and intranasal infection group.Hemorrhage in lungs and meningorrhagia in brain were found at 12 day post inoculation in intravenous injection group and intranasal infection group. swell and necrosis in spleen andhemorrhage in lungs were found at 15 day post inoculation in intravenous injection group and intranasal infection group. Comparing clinical signs, more serious lesion were found in intravenous injection group than intranasal infection group.(3) The primary microscopic lesions were congestion, hemorrhage, necrosis, massive infiltration of mononuclear cells consisting of lymphocytes, plasma cells, and histiocytes and lymphoid nodules. The lesions of brain showed a viral encephalitis change with perivascular space increased, encephaledema,glialnodule and massive microglia proliferation in brain parenchyma. The congestion of vessel, massive infiltration of mononuclear cells and massive inflammatory cells infiltration were found in the cerebral cortex at. The lesions of liver, kidney, pancreas mainly consisted of degenerate tubules with vacuolated epithelial cells, hyperemia and haemorrhage, and inflammatory cell disintegration. Spleen lesions contained hemorrhage, massive lymphocytes.Lesions of heart included cardiac muscle cells disruption, cardiac muscle fibers hemorrhage,massive infiltration by mononuclear cells, fragmetation of the myocardium and myocyte necrosis. And finally the lesions of lung included haemorrhage and pulmonary congestion.(4)The result of antibody levels showed that the antibody levels of serum of geese were rose gradually after infected with g AIV-H9N2. Then antibody levels reduced after reaching the peak. We can observe the antibody levels of intravenous injection groups rose faster and the value of preliminary antibody levels were higher than intranasal infection group. The most titer of antibodies were both 7log2 in intravenous injection group and intranasal infection group.(5) The test result of cell factor showed that the level of IL-2、IL-6、IFN-β、IFN-γ was higher than the control group obviously.(6) Total protein change trend: total protein level of geese in intravenous injection group and intranasal infection group rose gradually after infetion, reached its highest level after 6 day post inoculation, then decreased gradually. Total protein level of geese in intravenous injection group was higher than in intranasal infection group. Globulin trends: basic consistent with the change trend of total protein. AST/ALT change trend: after artificial infection, the level of the AST/ALT declined in geese in intravenous injection group and intranasal infection group.The change trend of creatinine:after artificial infection intravenous, creatinine level in intravenous injection group and intranasal infection group rose firstly and decreased after rising, reached to peak at 6 day post inoculation, and then decreased. The level changes in geese in intravenous injection group andintranasal infection group were almost the same, there is no difference.In conclusion, goslings in two-week old were sensitive to g AIV-H9N2. So in clinical process, we should pay more attention to the pathogenicity of g AIV-H9N2 for goslings, and do well operation for g AIV-H9N2 prevention and treatment. |
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