Analysis Of The Genotyping For Brucella Clinical Isolates Using Different Typing Methods

Bacteria of the genus Brucella are the etiological agents of brucellosis, which is a zoonotic disease endemic in many areas of the world. The disease will not only bring significant economic losses livestock, but also seriously endanger human health. The genus Brucella is divided into six classical species:B.abortus(bovine), B.melitensis(caprine and ovine), B.suis (porcine), B.canis (canine), B.ovis (ovine)and B.neotomae(ovly seen in the desert wood rat) on the basis of host specificity, antigenic differences and biochemical characteristics. B.abortus and B.melitensis are highly pathogenic and is a frequent cause of human brucellosis.In present,the typing methods of Brucella are phenotyping and genotyping. While genotyping has played an important role in rapid species identification, pathogen source analysis and differences between strains of Brucella.This study aims to find a fast,high-resolution typing method for clinical isolates of Brucella genotyping,analyze genetic polymorphisms of strains,establish phylogenetic relationships between strains,provide evidence for molecular epidemiology and pathogen source analysis of brucellosis in our country.23 Brucella clinical isolates were analyzed by 16S rDNA sequence analysis,AMOS-PCR, multiple locus sequence typing(MLST)and multilocus variable number tandem repeat analysis(MLVA) to Brucella genotyping,analyzing the characteristics of each method and to explore the relationship between the genetic evolution of clinical isolates.16S rDNA sequences of 23 isolates of Brucella after BLAST comparison showed that the sequences had the highest homology with Brucella, reaching more than 99%.And nucleicacid sequence similarity reached 99.85%.But 16S rDNA sequence analysis can not distinguish between species type of Brucella. Use of the AMOS-PCR assay for the species identification of Brucella showed that 22 of 23 isolates are B.melilensis biovarl,2 or 3,only 1 strain is B.abortus biovarl,2 or 4.To improve the resolution of MLST for Brucella.in the present study, we tested the feasibility of redesigning primers and improving resolution of MLST by increase of the sequencing length to 750bp,and established an extended MLST (EMLST). The valid sequences of product length amplified by traditional MLST(CMLST) primers are generally between 422bp to 589bp.While EMLST are between 611bp-759bp instead. To compare with the CMLST method,the nucleic acid length of all loci for EMLST were increased from 121bp to 302bp with the proportion increasing about 24.69%-71.56%.23 Brucella clinical isolates were analyzed with CMLST and EMLST.5 STs were defined by CMLST method,and 10 STs by EMLST.cobQ,glk,gap and ini-hyp of nine loci were at least increased one allele. Number of polymorphic loci is proportional to the number of genotype,polymorphic sites of four loci of nine genes were increased by increasing amplified product length.Cluster analysis showed that,whether genotype split decomposition analysis or evolutionary analysis, some of the strains that could be differentiated by CMLST were clearly differentiated by EMLST.It indicates that EMLST has a higher genotyping resolution than CMLST.27 STs were defined in 160 foreign Brucella strains from more than 30 countries by MLST analysis,and 3 STs were firstly defined in 23 Brucella isolates which had 5 STs defined-Named ST28, ST29, and ST30,ST28 were the advantages of this study ST type.PCR amplification and electrophoresis were used to distinguish in traditional MLVA method,16 highly polymorphic MLVA sites in 23 isolates were analyzed by this method in our study. The results of genotyping by MLVA-8 indicated that 22 were B.melitensis typed 115,only one was B.abortus typed 117.Genotyping by MLVA-11 and MLVA-16 had no results conforming to known genotypes in Genbank.The 16S rDNA, AMOS-PCR、CMLST、EMLST and MLVA data of 23 isolates showed that 16S rDNA sequence analysis is able to achieve the accurate identification of Brucella and have higher value on clinical diagnosis of Brucellosis,but not suitable for the species identification of Brucella.AMOS-PCR assay appears to be a rapid,simple and accurate diagnostic method of Brucellosis and also it can be used for identification of species and some biotypes of Brucella. EMLST which has a higher resolution than the CMLST can be used for study of epidemiology and flora polymorphism,and is suitable for Brucella strains of distant relatives.MLVA possessed the highest resolution of the study,which is suitable for Brucella strains of close relatives and epidemiology traceability.