Epidemiological Investigation Of Bovine Nodular Dermatosis And Isolation And Identification Of Epidemic Strains In Yunnan

In order to understand the occurrence and prevalence of bovine nodular dermatosis（Lumpy Skin Disease,LSD）in Yunnan Province,as well as the genetic variation characteristics of LSDV strains prevalent in Yunnan,the etiological and seroepidemiological investigation of LSD was carried out in cattle raised in some areas of Yunnan Province.At the same time,the isolation and identification of LSDV epidemic strains in Yunnan and the determination and analysis of the whole genome sequence were carried out.In this paper,LSDV antibody detection ELISA kit was used to detect 1270bovine serum samples collected from 8 prefectures and cities in Yunnan Province from 2018 to 2023,and LSDVq PCR and ordinary PCR detection methods were established to detect 2495 samples（including environmental samples,insect vector samples,blood samples,etc.）collected from 14 prefectures and cities in Yunnan Province from 2018 to 2023.The LSDV nucleic acid positive samples were analyzed by GPCR gene and P32 gene sequence alignment.At the same time,sheep testicular cells（ovine testicular primary cells）were prepared to isolate and identify LSDV Yunnan strains,and the second generation sequencing method was used to determine and analyze the whole gene sequence of LSDV Yunnan isolates.The experimental results show that:1.The total positive rate of LSDV antibody in serum samples is 34.33%（436 prime 1270）.The positive rate of LSDV antibody in Cunlan cattle in Yunnan Province is generally low,only in a large-scale field in Dali,the positive rate is 59.52%.2.In this paper,the Taq Man probe real-time fluorescence quantitative PCR and ordinary PCR detection methods for LSDV were successfully established,in which q PCR method can detect at least 4.83copies/μL of LSDV DNA,and the standard curve linear equation was established y=-3.755x+40.445,R²=0.9986,E=98.48%.The detection results of clinical samples show that the method is specific,sensitive,reliable and repeatable,and can be applied to the etiological detection of LSDV clinical samples.3.The total positive rate of etiology of collected samples was 0.24%（6 max 2495）.Among them,6 samples from cattle herds in 5states and cities were positive for LSDV nucleic acid,and the positive samples of LSDV nucleic acid were skin scab or festering liquid samples,but no samples of other visceral tissue disease materials,blood,environment and insect vectors（mosquitoes,ticks,moths）were detected.4.The results of amplification and sequence alignment of GPCR gene and P32 gene of LSDV nucleic acid positive samples showed that the homology of GPCR gene sequence of 6 LSDV Yunnan epidemic strains and 15epidemic strains at home and abroad was between 98.1%and 99.2%.The homology of P32 gene sequence with 13 LSDV epidemic strains in other regions was 99.1%and100%,and the homology with goat pox and sheep pox was more than 97%.The results of genetic evolution analysis of GPCR gene and P32 gene showed that Yunnan epidemic virus strain was closely related to Chinese LSDV-XJ-2019 strain,Thai YST strain and Russian Khabarovsk strain,and all of them were located in the same branch.The results of GPCR amino acid mutation analysis showed that compared with the reference strain LSDV-XJ-2019,there was a fourth point mutation in LSDV Nujiang strain,a fourth point mutation in LSDV Pu’er 1 strain,and a 346th point mutation in LSDV Qujing virus strain.The results of P32 amino acid mutation analysis showed that compared with the reference strain LSDV-XJ-2019,there was a mutation at site 8of LSDV Nujiang strain and a mutation at site 376 of LSDV Yuxi strain.No site mutation was found in other strains.5.A LSDV epidemic strain was successfully isolated from bovine skin scab samples collected in Yuxi,Yunnan Province,and named as LSDV-YNYX-2022 strain.Its TCID₅₀was 10^-5.875/0.1m L.The whole genome sequencing results showed that its nucleotide sequence was 145884nucleotides.The nucleotide sequence was compared with 10 LSDV reference strains at home and abroad,and the homology was 99.2%100%.The results of genetic evolution analysis showed that it was closely related to 9 domestic and foreign epidemic strains,and in the same branch,it was more closely related to LSDV attenuated vaccine strain Neethling strain and Russian Saratov strain.Compared with Thailand YST strain,there were 14042 point mutation（S mutation P）,44827 point mutation（H mutation P）and 44933 point deletion in the isolated strain.The results of this study can provide important theoretical data reference for the prevention and control of LSD and the screening of candidate vaccine strains.