Detection Of ADV,MEV And CDV In Mink Carcasses And Variation Of Isolates In Shandong Province And Safety Analysis Of ADV On Mice

Minamata is a high-grade fur economy animal,and it is currently based on artificial rearing.A large number of mink's carcasses have aroused widespread concern with the production of skin production.Because of the lack of scientific knowledge and processing technology for mink carcass composition at home and abroad,the carcass treatment of mink is very confused.Many farmers use mink's carcass to be used as raw materials for mink and other fur animals what brought significant biological safety risks to the fur animal husbandry industry.In order to clarify the status of viruses in mink's carcass and their safty in mice,this study established triples PCR detection method of Aleutian disease virus(ADV),Mink enteritis virus(MEV),and canine distemper virus(CDV),which were dangerous pathogens in minks.This rapid triple PCR method was used to detect these viruses from the main producing areas of Shandong.Variation analysis was performed on the isolated Aleuvirus strains and canine distemper virus strains,and the safty of ADV strain was analyzed in mice using artificial infection tests.The results are as follows:Three kinds of virus-gene-specific primers for MEV,CDV and ADV were designed and synthesized to optimize the reaction conditions.A triple PCR method for the rapid detection of three viruses was established.This method can simultaneously amplify the specificity of MEV 694bp,CDV 402bp and ADV 1025bp.The sensitivity test results showed that the minimum nucleic acid detection amount was MEV 3.13×10~5 copies/?L,CDV 1.94×10~5copies/?L and ADV 2.56×10~5copies/?L.The results showed that three virus PCR detection methods were successfully constructed for three viruses.The results of clinical samples showed that the results of three heavy and single PCR detection were consistent.It can quickly detect single or mixed infection in clinic.Using the triple PCR detection method,379 samples were tested and collected in the main breeding areas of 6 mink in Shandong province,and the results showed that the single infection rate was 2.9%to 8.6%.The single infection rate of MEV was 4.9%to 8.5%;The single infection rate of CDV was 5.1%to 6.3%;The mixed infection rate was 1.4%to 3.5%.The mixed infection rate of the ADV and CDV was 1.4%-3.4%.The mixed infection rate of MEV and CDV was 0.9%-4%.The infection rate of the three viruses was 0.9%to 2.1%.Isolation and identification of the ADV positive material were performed and homology analysis was performed.The results showed that a strain of ADV was successfully isolated and named ADV-SD.The strain was blindly transmitted on CRFK cells to the sixth day of the fifth passage and showed significant cytopathic effect(CPE).The comparison of the homology of the VP2 gene of the amplified ADV gene with the VP2 gene published at home and abroad shows that the homology of the VP2 protein gene of the ADV-SD isolate with other published strains ranges from 94%to 96%.The evolutionary tree was mapped to the ADV-SD-VP2 gene and it was found that the isolate and the domestic isolate were not in the same clade,but were in intermediate state of evolution between the domestic(accession number:GU183265-LN2)and Russian strains(accession number:KJ174161).The strains isolated from CDV positive samples were identified and analyzed by gene homology analysis.The results showed that a strain of CDV was successfully isolated and named CDV-SD;the strain had no obvious CPE(cell fusion was dewdrop)on Vero cells from the blind passage to the sixth passage,but a large number of cell death was observed;The H protein gene homology analysis of the isolate showed that the strain was as high as 99%in consistency with the published Shandong reference strain.The phylogenetic tree was mapped to the CDV-SD-H gene and it was found that the isolates were in the same clade as the three domestic strains in Shandong(accession numbers of Shandong isolates were KF880682,KF880683,and KF880684).The results of ADV isolates showed that 8 mice in the test group were infected with ADV,but there was no significant difference between the body weight and the visceral organ development index of the infected mice,and there were no obvious pathological changes.The blood routine test showed that the total number of lymphocytes in the infected mice and its ratio increased significantly(P<0.05),the total number of platelets and the grain size were fine.The total number and ratio of cells and the ratio of intermediate cells decreased significantly(P<0.05),and the difference of other indexes was not significant(P>0.05).The antibody titer of the influenza strain was significantly lower than that of the control group(2~3vs 2~4,p<0.05);the ADV was isolated and identified in mice,and the homology of the VP2gene was compared.The results showed that the ADV strain was carried in mice.The homology with the original strain was 99.2%,indicating that ADV can infect mice and show slight variations.Through the above studies,the established triple PCR method for immediate detection of ADV,MEV,and CDV provides important methodological basis for understanding the status of three types of viruses in leeches and their carcasses,clinical epidemiological investigations,and differential diagnosis;and identifying leeches in the main producing area of otters in Shandong Province.The evolutionary relationship between the tested strains was evaluated,and the pathogenicity of ADV isolates to mice was evaluated.The results provide a scientific basis for understanding the biological safety of mink's carcass and exploring the criteria for the classification and utilization standard of mink carcasses.