Development Of Immunohistochemistry And RT-PCR Dot Blot Assay For Detection Of Avian Influenza Virus

12 four-week old SPF layers were divided randomly into two groups, six for the tested group and six for the control group. Each layer of the tested group were inoculated H5N1 pathogenic avian influenza subtype virus allantoic fluid of poultry embryo by intranasal routes. Each layer of the control group were inoculated equal non-bacteria allantoic fluid of poultry embryo by intranasal routes. All tested group deaded after 40 hours inoculated, killed all layers, took tissues and fixed these tissues in 4% paraformaldehyde. The paraffin section was prepared and then H.E.staining and Immunohistochemistry detection were carried out. The clinical symptom showed that the tested layers were dispirited deeply, began to eat little after 24 hour inoculated. Kidney had a change of swelling. Petechchiae and hemorrhage appeared in intestines mucosa. Pancreas were hemorrhage. Bursa of Fabricius became atrophic. Gizzard and proventriculus were hemorrhage. H.E.staining results showed that the most pathological changes were congestion in kidney, denaturation and necrosis of epithelial cell of renal tubule. The lymphocytes are necrosis and the number is reduce in spleen, bursa of Fabricius, thymus and cecum tonsil. Congestion appeared in lung and its epithelial cell were denaturation, defluvium defluxion. The mucous epithelial cell of larynx were denaturation, defluvium defluxion. Liver were hemorrhage, its cells were swelling and denaturation. There were hemorrhage, denaturation,necrosis in intestines and Pancreas. Immunohistochemistry staining results showed that there were positive cells in kidney, sleep, thymus, cecum tonsil, lung and larynx. Especially in kidney and lymphoid organizations. The avian influenza virus strain invaded epithelial cell and lymphocyte, and existed in cell membrane, cytoplasm and karyon of infected cells. There was not positive cell in the control tissues.40 four-week old SPF layers were divided randomly into two groups, twenty for the tested group and twenty for the control group. Each layer of the tested group were inoculated H9N2 subtype low pathogenic avian influenza virus allantoic fluid of poultery embryo by intranasal routes. Each layer of the control group were inoculated equal non-bacteria allantoic fluid of poultry embryo by intranasal routes. Killed randomly 4 layers of the tested group and 4 layers of the control group after 1d,3d,7d,14d,21d inoculated. Took tissues and fixed these tissues in 4% paraformaldehyde. The paraffin section was prepared and then H.E.staining were carried out. The clinical symptom showed that the tested layers were dispirited deeply, began to eat little and drunk more, and some layers'ordure watery after 3 day inoculated. Kidney had a change of swelling severe. Liver became swelling, hemorrhage and appeared serosity exudation. Swelling and hemorrhage appeared in intestines mucous.H.E.staining results showed that the most pathological changes were congestion in kidney, denaturation and necrosis of epithelial cell of renal tubule. The epithelial cell of intestines mucous were denaturation, necrosis and defluvium defluxion. Lung were hemorrhage. The mucosa of trachea were denaturation and deprived. Liver were hemorrhage, congestion, its cells were denaturation. The lymphocytes are necrosis and the number is reduce in spleen, bursa of Fabricius, thymus and cecum tonsil.Based on sequences of HA gene of H5N1 and H9N2 selected in GENEBANK, we designed primers. We developed RT-PCR methods to detect H5N1 and H9N2 subtype avian influenza virus by the two pair of primers. We used the other two pairs of primes to amplify two fragments of HA gene and labeled as probes by DIG. Through a dot blot experiment detected productions of RT-PCR of avian influenza virus RNA by probe, it developed the RT-PCR dot blot method. The results showed its sensitivity was 100 to 1000 times as much as that of RT-PCR method.