Identification Of Sheep Maedi Visna Disease In Inner Mongolia And Establishment Of PCR Detection Method

Maedi visna disease(MVD)is a multi-organ injury-transmitted disease caused by Maedi visna virus(MVV).The disease can cause diseases in the brain,lungs,breasts,joints,etc.,and once infected,it eventually causes death.MVD seriously affects the development of the sheep industry and gives the sheep industry a serious economic loss.In order to identify the presence of MVD in Inner Mongolia,12 lung disease tissues were collected from a slaughterhouse around Hohhot for histopathology and PCR identification.Histopathological results showed that 3 lung tissues were fibrotic,and type Ⅱ alveolar epithelial cells proliferated and interstitial widened in the alveolar cavity.More lymphoid follicle hyperplasia was observed around the bronchi and bronchioles.PCR results showed:Three lung tissues were PCR positive,and the sequencing sequence was 87.4%homologous to the MVV KJ641278 US strain registered in GenBank.To further determine that 3 lung tissues do have MVV infection,the LTR according to the full sequence of MVV(accession number KJ641278)A pair of primers was designed in the region,and 3 lung tissues were positive for MVV.The presence of MVD in Inner Mongolia was determined by histopathology and PCR identification.In order to establish a MVV-specific nested PCR assay,nested PCR primers were designed and synthesized according to the MVV(Accession No.KJ641278)gene sequence published on GenBank,and nested PCR was performed on the collected lung tissues.The results showed that 3 positive MVD disease lung tissues could be detected,and the sensitivity test could detect at least 0.894 fg of viral DNA.The specific test results showed no cross reaction with P.falciparum,M.pneumoniae,Pneumococci,and Pasteurella..In order to explore the viral load of MVV in diseased lung tissue,the positive plasmid of MVV was used as a template for quantitative PCR amplification,and the amplification curve and standard curve were obtained.The quantitative PCR method was established.The results of fluorescent quantitative PCR showed:It is capable of specifically detecting 3 copies of MVD-positive lung tissue and detecting a minimum of 2.74×102 copies/μL of viral DNA.In order to identify whether 15 peripheral blood leukocytes collected in the clinic were infected with MVV,this experiment used nested PCR detection to detect it.The results showed that 8 out of 15 peripheral blood leukocytes were positive for MVV.Because MVD and Ovine pulmonary adenomatosis(OPA)often occur mixed infection,in order to identify whether the collected lung tissue and peripheral blood leukocytes are also infected with OPA,this experiment uses PCR,nested PCR and combined diagnostic PCR.Perform test analysis.The results of common PCR showed that all 3 MVD positive lung tissues were positive for OPA,and the sequence homology was 100%between the sequencing sequence and the JSRV gene sequence registered in GenBank(accession number JQ837489).The nested PCR test results showed:15 copies.Ten of the peripheral blood leukocytes were positive for JSRV;the results of combined diagnostic PCR showed that MVV and JSRV were positive in 3 diseased lung tissues.The above results indicate that there is MVD infection in Inner Mongolia,sometimes accompanied by mixed OPA infection.The nested PCR detection method established in this experiment can be used for the specific detection of MVD,and the real-time PCR can be used for the detection of viral load of MVV.Both detection methods have the advantages of good specificity and high sensitivity.Early diagnosis provides a convenient,economical and practical detection technology,which laid the foundation for the study of MVD in Inner Mongolia.