| Canine distemper（CD）is an acute and highly contagious disease which was caused by thecanine distemper virus （CDV）of paramyxoviridae and morbillivirus. The CDV can infect avariety of animal, and the mortality rate may reach as high as80%. Therefore, the CD is known asthe "devastating infectious disease". When the CDV infect dog, mink or fox, the mortality rate is30%to80%, but it can reach as high as100%in ferret.Tthe range of CDV naturally infectedanimal is expanding and its harm is also growing.CDV may proliferate in not only pulmonary macrophage, peritoneal macrophages and renalcells of canine, but also kidney cells of calf and chick embryo fibroblast. But the caninepulmonary macrophages are most susceptible to CDV, and it can display the typical thyroids celllesions（CPE）after CDV infected. However, the separation of canine pulmonary macrophages isdifficult and it is easy to pollution. The CEF can stably grow on the low nutritional medium and itis sensitive to CDV. The CPE is CEF typical and easy to observe after the CEF infect CDV. Butthe first separation of CDV in CEF is very difficult because the CDV may adapt to CEF aftermultiple passages in the chick chorioallantoic membrane（CAM）. In recent years, people foundthat the CDV possess the lymphocyte stimulation factor（SLAM）and the Vero cells may expressSLAM receptor. Therefore, the Vero cells become the preferred cell to isolation and culture ofCDV. In the research, we selected the CDV positive samples of mink after RT-PCR detection inthe veterinary diagnostic center of Lubei and inoculated the CEF, DF1and Vero cells respectivelyafter routine process. The Vero cells were defined as the optimized cell to CDV after contrastedthe sensibility of every cell. The Vero cells finally appeared the typical CPE after CDV infectedafter the optimization of cell culture conditions. The CDV was harvested and identified byRT-PCR detection, SN test, IFA test and virus inclusion body examination. The results showedthat the CDV was positive and was separated successfully. It will provide the material for theresearch of biological products, the establishment of related detection methods and the effectiveprevention and control of CDV.The main detection methods include the neutralization test, ELISA, immunofluorescenceassay, colloidal gold test strip method. These methods possess the advantages of sensitivity,specificity, accuracy than the conventional virus isolation and culture. However, the technology ofPCR has all these graces, but also it may provide enormous helps for the diagnosis in the earlystage of CDV infection. The CDV is a single strand and unsegmented RNA virus. In structuralproteins of CDV, the N protein is most and processes the strongest conservatism asimmunogenicity protein. It is the main component of the nucleocapsid of CDV. The N protein isgreatly significant for the early diagnosis of CD because it can induce the early immune responseafter CDV infected. Therefore, we established a real-time fluorescent quantitative RT-PCRtechnique for the N protein of CDV based on RT-PCR detection methods. The result will providebasis for the early diagnosis of CD and the effects of antiviral drugs and vaccines inoculation. In order to obtain the higher titer of CDV, the culture conditions of CDV on Vero cells wereoptimized through the application of the technology and the determination of TCID50.The vaccinal inoculation is the only effective prevention and control measures of CDVbecause there is no specific treatment at present. The conventional vaccines include inactivatedvaccine and attenuated vaccine at home and abroad. The inactivated vaccine can induce hum oralimmune response only. Although the attenuated vaccine can induce hum oral and cellular immuneresponse, it has quite poor thermal stability and it is easy to be interfered by its maternal antibody.Furthermore, the virulence of the attenuated vaccine is too strong for some fur animals. Therefore,the domestic and foreign scholars devoted to the development of the more safe and effectivevaccine, the hyper immune serum and the yolk antibody（IgY）to control CD more effectively inrecent years. In the research, the CDV vaccine was prepared after the CDV was inactivated andemulsionized with Freund’s adjuvant on the basis of the isolation and culture of CDV. Sequently,the hens were inoculated with CDV vaccine many times and the CDV refined yolk antibody wasprepared. At the last, the animal test was progressed to detect the protection efficiency of IgY.The result showed that the CDV refined yolk antibody is good. Therefore, the better treatment ofIgY may provide reference for the research of the CDV related biological products and theeffective control of CDV. |
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