| The study isolated the canine distemper virus(CDV) named CDV-1 in Vero cells from the dog which was infected by CD in clinic after final diagnosis.The CPE in Vero cell appeared on the stability after generation cultivation in gathered shedding,rounding, net-balloon features.The globular virion could be seen in cytoplasm with 150nm size by electron microscope.In physics and chemistry experiments,the virus was inactivated 56℃for 30min,more sensitive to changes of acidity.In pH 7.0-8.0 environment virulence held stability but sensitived above pH 9.0 and below pH 6.0.The other feature of CDV-1 is that containing peplos,could not agglutinating red blood cell of chicken,rabbit and sheep.The TCID50 of CDV-1 is 10-2.13/ml,10-4.81/ml,10-5.35/ml and 10-5.832/ml high after cultivated 5d, 7d,9d and 10d in order.The serum neutralization test finally proved that the serum can neutralize the virus to absorbing on the cells,and the positive serum titer was 1:13.In animal tests,the 1×105.8 TCID50 1ml,3×105.8 TCID50 1ml and 6×105.8 TCID50 1ml density gradient was chosen to inject in abdominal cavity of dogs.Among the total,the dogs injected 6×105.8 TCID50 1ml were appeared a clinical typical symptoms.The tested dogs appeared symptoms in 4-7 days,appetite was disappeared,catarrhalic rhinitis, diarrhea,purulence discharge in eyes and body temperature presented diphasic fever,at last died exhaustion.After biopsy,apex of lung hyperemia and aerocele,liver congestion and its texture became fragile,lymph node intumesce,seroperitoneum,the intestine mucosa hyperemia and fallen off and the edge of spleen infracted.The epithelial cell of mucous membrane of urinary bladder were scraped and dyed by hematoxylin-eosin,after that,the red bodies were seen in cytoplasm.Liver congestion and the liver cells became vacuole, alveolar wall became thickening,and phlegmasiaed cells collected in trachea,many silver-staining phil bead in spleen,lymphocyte cell population reduction of Spleen white pulp could be seen in pathological section.The appetite of two dogs which injected 3×105.8 TCID50 1ml density was disappeared and spirit depressed.After 7 days injection,the dogs' temperature rised slightly and the dogs live also.The dogs which injected 1×105.8 TCID50 1ml density were appeared symptoms not obvious,and the dogs which injected nothing were appeared normal.In our study,the immunogenicity of inactivate viral antigen was compared with live viral antigen,both of them were made from CDV-1.inactivate viral antigen(the density was 1×105.8 TCID50/ml),immuned two times,the first time and the second time intervals three weeks,each time 2ml,and conteracted toxic substances 5×105.8 TCID50/1ml after 28 days from abdominal cavity to nulli-inoculate dogs,compared with live viral antigen(the density was 5×103.8 TCID50/ml and 5×102.8 TCID50/ml immuned one time,each time 1ml. Collected the serum of 5d,10d,20d,27d after immune,of 7d,12d after attacking toxin and detected the serum by indirect ELISA,the antibody changed regularity could be known.The result showed that the dog injected inactivate viral antigen has produced effective immune response,after 21 days and 28 days immuned,the antibody level reached 0.46 and 0.49,then tendency to stabilization.The antibody level of the dog injected live viral antigen is lower totally than the dog injected inactivate viral antigen(0.01＜P＜0.05), and with the density of 5×103.8 TCID50/ml is higher than the density,of 5×102.8 TCID50/ml, but the disparity is not predominance(P＞0.05).The antibody level of the dog injected inactivate viral antigen is raised rapid after the second immune and reached the highest after two weeks of sceond immune.Both of the antibody level is raised slowly,and the antibody level reached 0.3 only after four weeks immunity.The antibody level of the dogs was lower than injected virus before,and also the antibody level of dogs which were injected canine distemper virus was lower obviously than the dogs without of canine distemper virus in the same group synchronization. |
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