Genetic Polymorphism Analysis Of Brucella Melitensis Isolates From Northern China

Brucellosis is a serious zoonotic disease that exists in more than 170 countries and regions.Only a few developed countries have announced that they have eradicated brucellosis.In this study,51 strains of B.melitensis isolated from some provinces in northern China during 2010-2016 were studied.1.Typing by traditional biochemical methods.The results showed that most isolates were identified as B.melitensis type 3,accounting for 92% of the total of isolates,followed by the B.melitensis type 1 and type 2.2.Establishment of MLVA-15 genotyping method.The genome senquence of reference strain 16 M was analysed using on-line software(http://tandem.bu.edu/trf/trf.html)to search the VNTR sites.Out of the 95 VNTR loci,47 VNTR sites were selected based on 100% match standard of tandem repeat sequence.Of which,25 were located on chromosome No.1,and 22 on chromosome No.2.After analysis of HGDI index,15 VNTR sites were finally selected to establish MLVA-15.8 VNTR sites were the same as previous MLVA-16,and 7 highly polymorphic VNTR sites were newly selected.Analysis by software BioNumerics 7.6 clustering classification showed that the differentiation effeency of domestic Brucellaisolates by MLVA-15 was significantly higher than MLVA-16.The basis lies in that,The MLVA-15 reduces the VNTR sites showing no polymorphism in MLVA-16 and improves the discrimination ability of the epidemic isolates of Brucella,ensuring the exact trace back needs of infectious sources and transmission routes in the epidemiological survey of brucellosis.3.Multi-locus variable tandem repeats analysis(MLVA).Based on MLVA-8 analysis,the majority of Brucellaisolatesfrom China were type 42,accounting for 70.6% of the total isolates,followed by Type 63 and 43;2 new genotypes were found.Based on MLVA-16 analysis,a total of 28 genotypes were found,reflecting the higher genetic polymorphism of B.melitensis in China.The minimal phylogenetic analysis proved that the B.melitensis isolates from China were genetically most closeed to the strains in Mediterranean countries,revealing the main spreading path from the Mediterranean to China in the long history.4.Multi-locus sequence analysis(MLSA).The MLSA-21 analysis showed that 37 strains of B.melitensis belonged to ST8.3 new genotypes were found,including ST137(1 isolate),ST138(1 isolate)andST139(12 isolates).This method showed highly reproducibility and standardization.5.The whole genome single nucleotide polymorphisms analysis(WGS-SNP).Based on WGS-SNP analysis,the isolates of B.melitensis that are currently endemic in China belonged to the Eastern Mediterranean lineage.Phylogenetic analysis using Mega7 software showed that the prevalent strains in China are closely related to isolates from Mediterranean countries such as Turkey and Kuwait,as well as to Asian countries,and are clustered with the reference strains of B.melitensis type 2.