| Canine distemper virus(CDV) belongs to genus Morbillivirus of Paramyxoviridae which can cause animal diseases such as marten,fox,raccoon dog,dog,panda and tiger. This disease has always been focussed on researching by researchers all over the world because its high infectivity,high morbidity and high mortality,and also its relationship with subacute sclerosing panencephalitis(SSPE) and multiple sclerosing(MS) of human. However,there is not a lot of systemic research on the etiology,immunology,pathology, diagnostic techniques and new technique of vaccine.During the period of June to October in 2006,this disease broadly broke out in fur-bearing animals in Shandong,Hebei,Jiangsu and Liaoning provinces.Here,we isolated and identified 5 CDV isolates from raccoon dogs, foxes and minks after observation of the pathology of the clinical ill animals.Then we analysed the molecular biological properties of the 5 isolates and studied on the immune efficacy of four recombinant plasmid DNAs expressing the H,F and N gene of CDV.1.Study on the clinical pathologySuspicious CD epidemic raised in part fur-bearing animal farms in Shandong,Hebei, Liaoning and Jiangsu provinces from June to October in 2006 was observed and analysed from epidemiology,clinical situation and pathology.Then,we studied on the pathology of selected samples.The results showed that this CDV epidemic was mainly caused by allocate and transportation of raccoon dogs and minks carrying CDVs.Pathological lesions were characterized as haemorrage of whole body tissues,and its severity level from high to low was raccoon dog,fox and mink,respectively.Pathology comparison of lung,liver, brain,kidney and bladder indicated that there was no obviously difference apart from the pathological changes between minks and fox/raccoon dogs.However,pathological lesion difference existed in the same species animals according to disease of course,type of disease and complications. 2.Isolation and identification of CDV isolates,and research on their biological propertiesBased on the observation of epidemiology,clinical signs,pathological changes and histology,several viruses were isolated from samples of liver,spleen,lung,lymph node and brain of ill raccoon dogs,foxes and minks,respectively,by using Vero-DST cells expressing dog's SLAM,Then the isolates were identified by by electron microscope with phosphotungstic acid(PTA) negative staining or ultrathin section of cell cultures,specific nuclear acid detected from infected vero-DST cells by RT-PCR,detection of physical and chemical characters,and artificial infecting animal tests.The result showed that clearly CPEs were observed on Vero-DST cells at 36-96h after inoculated with treated samples from 5 different origins.TCID50 of the 5 isolates was 10-5.2～-7.3/ml.Multi-morphous virion about 110～500nm large was observed by electron microscope.All the 5 isolates were positive detected by specific RT-PCR for CDV.All the isolates were not observed hemagglutination with red blood cell(RBC) except weakly hemagglutinin of chicken and goose RBC.The isolates were sensitive to diethyl ether,chloroform,formaldehyde,and weak power of resistance to acid,alkali and high temperature.LD50 of 5 isolates to suckling mouse was 2×10-3.8～-4.8/ml.They all had powerful pathogenicity to raccoon dogs,but no pathogenicity to experiment rabbits.According to the above results,all the isolates were identified as CDV and named as CDV/SD/RD06/HT-P(raccoon dog), CDV/SD/RD06/THDI(raccoon dog),CDV/SD/M06/HD(mink),CDV/SD/M06/LN(mink) and CDV/SD/F06/HB(fox),respectivey.3.Sequence analysis of H and N protein gene of isolates from mink,fox and raccoon dogThe H and N genes of 5 CDV isolates were amplified and cloned into PGEM T-Vector.The sequences of all these H and N genes were compared with other sequences published in GenBank.Results revealed that:(1) the homology of H gene nucleic acid sequences among the 5 isolates reached more than 97.5%,however,those among the isolates and vaccine strain chn was 90.5-91.8%.Amino acid homology between HT-P and THD1 was 100%,but had great distinction with the other strains,87.9-99.1%.The deduced aa sequences of the 5 H genes were generally low compared with chn,89.7%-91.8%. However,there was high homology between chn and Onderstepoort or Convac with nucleic acid homology of 96.8%and 97.2%respectively,and amino acid homology of 97.1%and 95.6%respectively.(2)the homology of N gene nucleic acid sequences among 5 isolates was more than 94.5%,with highest homology between HT-P and THD1,99.8%.HD origin site was far from LN's,but they had higher homology,however,it had relatively lower nt homology to HB.This result might hint that species differences existed between field isolates when they infected susceptive animals.Generally lower homology of nt sequences were observed between elan and isolates,90.9%-93.5%,but there was higher homology of nt between chn and Onderspoort,97.1%.On view of aa sequence of N proteins,there was lower homology between isolates except HT-P,THD1 and HD.Chn had very high homology with Onderstepoort,with their homology reaching 97.3%.These results showed that there was powerful difference of immunocity both H and N protein with vaccine strains, which might be the key factor of CD failure immunization.4.Study on rapid test method for CDV by Real-time TaqMan RT-PCRSpecific primer&probe were designed and synthesized according to CDV N gene. After optimization,a Real-time TaqMan RT-PCR was established.Powerful fluoescence signal and smooth curve were gained with 20pmol/mL primer concentration 1uL and 20pmol/mL probe concentration 0.3uL.High sensitivity,with testing RNA concentration as low as 1.24×10-3ng/uL;High specificity,with no cross reaction to NDV,AIV and NiPV. Coefficient variation(CV) of test repeatability was 2.3%,2.5%and 4.2%,respectively.In order to examine the correspondency level among new established menthod and routine RT-PCR,BIOINDIST BIT RAPID CDV kit,57 clinical suspicious CDV samples were detected with three above-metioned methods.Results revealed that positive rate was 93%(53/57) with established TaqMan RT-PCR method,while BIOINDIST BIT RAPID CDV kit and routine RT-PCR was 70.2%(40/57) and 71.9%(41/57),respectively. Coinfidence of TaqMan RT-PCR to BIOINDIST BIT RAPID CDV kit was 77.2%,and to routine RT-PCR.was 78.9%.However,there was high correspondency level between BIOINDIST BIT RAPID CDV kit and routine RT-PCR with their coinfidence 98.2%. Results showed that the Real-time TaqMan RT-PCR,developed in this study,had good characters of rapid,specificity,sensitivity,and could tested large quantity of samples at the same time.It could be used in early detection of CDV infection and quarantine at customs.5.Construction and identification of recombinant plasmid expressing H,F and N gene of CDV isolated from raccoon dogsThe H,F and N(N1,N2) genes were amplified from CDV RNA by RT-PCR, respectively,and cloned into pMD18-T vector.After sequence determined and analysed, these gene were sub-cloned into pIRES1neo expression vector,respectively.The recombinant plasmids DNA were transfered into VERO-DST cell,respectively.Results of Western-blotting showed that aim protein gene could be expressed and used to study on the immunogenicity of CDV genes in animals.6.Protective efficacy of recombinant plasmids expressing CDV H,F and N gene in raccoon dogIn order to observe immune reaction of the recombinant plasmids pIRES H,pIRES F, pIRES N1 and pIRES N2,fourty-two 16-18g body weight experimental white-male-mice were randomly assigned to seven groups each with six.Group 1 was vaccinated posterior limb intramuscularly multi-site injection with pIRES H and boosted two times with 2 weeks interval.Group 2,group 3,group 4,and group 7 was vaccinated pIRES F,pIRES N1 + pIRES N2,pIRES H+ pIKES F+ pIRES N1 +pIRES N2,pIRES empty vector, respectively.Immunization route and inoculate dosage were the same as group 1.Group 5 was vaccinated with live attenuated vaccine which was used broadly in fur-bearing animal farms in China.Group 6 was injected subcutaneously with CDV-CPV imported vaccine according to the instruction.Group 5 and group 6 were also boosted two times after first immunization.Each group was blooded before first immunization and each boosting immunization by cutting tail of the mice.Two weeks after the last boosted,blood was collected from mice by removal their eyeballs.When serum was isolated from the blood, ELISA and neutralization antibody titers were detected respectively.The results showed that high level of CDV-specific ELISA antibody and neutralizing antibody could be induced after two boosted vaccination compared with pre-vaccination and group 7(p＜0.05). Compared to ELISA antibody titer,the difference between group 4 and group 1,2,3 was not significant,but its neutralization antibody titer was obviously higher than single DNA vaccine(p＜0.05).Neutralization titer distinction between group 5 and group 4 was not significant,but its ELISA titer manifest higher than group 4.Group 6 was obviously higher than any of the gene groups both ELISA and neutralization titers.To furtherly assess the immuno-efficacy of the above recombinant plasmids against CDV,twenty-one 45～60-day-old raccoon dogs(NT≤1:2) were assigned to seven groups each with three.Each group was treated as the above mice immunization test.Two weeks after the last boosted,each group was oronasally and intraperitoneally challenged with virulent CDV isolate THD1.Results revealed that high level of CDV-specific ELISA antibody and neutralizing antibody could be induced after three times vaccination.After challenged,raccoon dogs in vaccinated group pIRES H,pIRES F,pIRES N1 + pIPES N2 had different level mortality,except Group pIRES H+ pIRES F+ pIRES N1 +pIRES N2.No raccoon dog died in Group CDV-CPV imported vaccine,and live raccoon dogs were also showing slighter signs than those of other groups.Clinical signs and pathological lesions in single gene vaccine vaccinated-challenged group was similar to each other,while slighter than that in empty control group.But it was significantly high compared to Group pIRES H+ pIRES F+ pIRES N1 +pIRES N2 and Group CDV-CPV imported vaccine(P＜0.05). Meanwhile,virema presented in vaccinated group were milder than those in control group. It indicated that the recombinant plasmids were able to confer significant protection against clinical diseases and reduce pathogenic lesions induced by virulent CDV challenge,even though it could not provide complete virological protection and could not resist clinical signs development.The recombinant plasmids might be an attractive candidate vaccine for preventing the disease associated with CDV infection.
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