| Canine distemper(CD) is an acute and highly contagious disease by the canine distemper virus.The clinical typical characteristics include anorexia, bipolar fever,conjunctivitis, severe gastroenteritis and neurological symptoms. Canine Parvovirus(CP) is an acute contagious disease by the canine parvovirus(CPV). The clinical typical characteristics include hemorrhagic enteritis or non-suppurative myocarditis. The susceptible host range of CDV and CPV continues to expand, spreading in a large number of species between intra- and interspecies. In recent years, giant pandas infecting CDV and red pandas infecting CPV have been reported. There are no specific immune serums and corresponding vaccines against CDV and CPV currently.In this study, PCR and gene sequencing, cell culture assays, animal virus regression tests,viral inactivation testing, immunizations, and test methods etc.were utilized.The CDV of giant panda breeding base in Shaanxi Louguantai were investigated and the CPV of Fuzhou Giant Panda Panda were investigated. CDV from gaint panda and CPV from red panda were isolated and identified. Canine parvovirus inactivated vaccine and immune effects assessment were carried on. The results can be concluded as follows:1.This study successfulyl diagnose Canine distemper of the giant panda breeding base in Shaanxi Louguantai distemper epidemic by PCR. CDV of anticoagulant detection rate was16%, cotton swab detection rate was 28%.Heart, lung and liver samples from a Giant Panda dead of Canine distemper(CD) were collected in the present study. After grinding and repeated freezing and thawing, the supernatant was inoculated on Vero cells, and the cell cultures were collected after repeated freezing and thawing when cytopathic effect(CPE)appeared. Isolated virus was identified by reverse transcription polymerase chain reaction(RT-PCR) and animal regression testing, and the titer was detected by the method of Reed-Muench. The results suggested that CPE, including cell aggregation, round shrinking,network and pull off the ultimating, appeared after blindly passaged for five generations. ThePCR product was 287 bp which was consistent with expectations and was sequenced, and the isolated strain showed an identity of 98% with the CDV strain SD(14)11(Asia-1) at the level of nucleotide. The titer of the isolated strain was 10-5.2TCID50/mL. Puppies appeared CD clinical symptoms after challenging the isolated CDV. In conclusion, the Giant Panda Canine Distemper Virus of LG strain was successfully isolated.2. In general CPV detection rate was 10 % in peripheral blood and cotton swabs of red pandas in Fuzhou Panda World. After pretreated, samples were detected by PCR,and inoculated on F81 cells.The F81 cell cultures with CPE were colleted and identified by PCR,and used to challenage yang canine.The results show that, blood samples and rectal and saliva swabs were CPV positive, and CPE appeared in third F81 cell blind passage, and the PCR products sequenced were CPV.The challenaged canines shows typic symptom of canine parvovirus disease.The results indicated that a CPV strain, which may cause canine parvovirus disease in red pandas, were isolated successufly,named CPV FZ strain.3. CPV was amplificated by cell culture and inactived by formaldehyde. The TCID50 of CPV was 10-5.6/50 μL. Inactivated CPV vaccine develop with adjuvant that were transfer factor and aluminum salt adjuvant gel.The immune seras antibody titer achieved 1: 28.6after three time immunity.Dogs did not appeare clinical symptoms, and the antibody titer was1: 211.Conclusion: The CDV LG virulent strain and CPV FZ virulent strain were successfully isolated.The method of CPV Inactivated vaccine and immunization program were estibaled.Canine parvovirus inactivated vaccine assesses good results. Corresponding inactivated vaccine and seras were achieved. The results provide a reference for the giant panda and the red panda to against CDV and CPV. |
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