Molecular Epidemiology Of Sheep Brucella In Northwest China And Screening Of Antigenic Epitopes

Brucellosis,causing by Brucella spp.that is a facultative intracellular gram-negative bacteria,is a common zoonotic disease that affects both humans and animals.In recent years,brucellosis has re-emerged in China,resulting in zoonotic spillover to humans and serious economic losses.This work aimed to analyze the molecular epidemiology of Brucella in northwest China,and to understand the dominant Brucella strains and its genetic characteristics,tracking the origin and variation of the epidemic strain.According to the genomic characteristics of the popular strains,the conservative immune-antigenic proteins were used to screen antigenic epitopes.A total of 480 spleens from the aborted fetuses of sheep and yaks were collected in the northwest region of China,including Gansu,Inner Mongolia,Xinjiang and Qinghai provinces.66 isolates of Brucella were recovered from sheep and yaks.Using classical biotyping and the Brucella AMOS PCR assay,all isolates were identified as B.melitensis biovar 3(n = 58),B.melitensis biovar 1(n = 1),B.abortus(n = 5),or B.suis biovar 3(n = 2),and B.melitensis biovar3 was found to be mainly responsible for sheep brucellosis in northwest China.MLVA-16 identified 46 genotypes in these populations,including 37 unique and 9 shared genotypes,and the HGDI is 0.979.MLVA-11 showed that 71% of the isolates(49/66)were genotype 116(1-5-3-13-2-2-3-2-4-41-8),a characteristic subgroup of the East Mediterranean group,showing that isolates from different geographical areas exhibit similar epidemiological characteristics in different regions and may be epidemiologically linked.MLVA-11 also revealed that an isolate from Inner Mongolia had a novel genotype,genotype 369(1-5-3-13-2-2-3-2-7-41-8).This study is the first analysis of B.melitensis in Gansu province,and the results confirmed that in this province,isolates of this species are disorderly and unsystematic.MLST result showed ST8 is a dominant genotype among those B.melitensis isolates.In addition,a large-scale survey was conducted in domestic animal populations from 2011 to 2015 in Qingyang,China.From 2011 to2015,the average positive rates in every year were among 0.04% to 4.75% in the eight counties,and the prevalence rate was among 0 to 9.96% in eight counties.The results of serum epidemiology confirmed that the region of northern Qingyang was critically Brucella epidemic areas and the disease was attaining a rising trend,especially from 2013 to 2015.Analysis of genotypes of isolates suggested that sheep brucellosis was mainly resulted in conventional B.melitensis(East Mediterranean group),and the external strain(America group)was also appeared in Qingyang.Two B.melitensis strains,including B.melitensis QY1 isolated from Gansu and B.melitensis QH61 isolated from Qinghai respectively,were carried out to sequence and analyze of the whole genome.The results showed that the genomes were highly conserved between Brucella species.The sequenced Brucella strain has two circle chromosomes with 3.3Kb at length.The whole genomes size of B.melitensis QY1 and B.melitensis QH61 were predicted 3093 and 3057 encoding genes,respectively.Phylogenetic analysis revealed close relationship and highconsistence between Brucella isolates from China.Analysis of dispensable genes showed that high differences were presented in genomes.Based on analysis of the genomic characterization of 66 isolates,immunogenic Omp16 and L7/L12 proteins were choice for screening potential antigenic epitopes,and a total of 10 probable antigenic epitopes were predicted by bioinformatics tools.Epitopes were assessed using dendritic cells(DCs)by detecting different cytokines released level in cultural supernatants after epitopes were stimulated DCs.The selected epitopes were evaluated in BABL/c mice.The results showed that omp16-1 and L7/L12-4 were T cell epitopes,which could stimulate cell and immune animals to play the cellular immune function.Omp16-5 can have a strong reaction with the anti-Brucella serum,and can also play the humoral immunity function.In summary,our results demonstrate that sheep were main infected B.melitensis bv3 with the characterization of genotype 116(MLVA)and ST8(MLST).Epidemic strains were derived from East Mediterranean group and highly conserved in genome.The application DC can effectively screen the epitope,and successfully screened good epitopes from Omp16 and L7/L12 protein.