ABSTRACT
Mongolia has a high incidence of brucellosis in human and animals due to livestock husbandry. To investigate the genetic characteristics of Mongolian B. melitensis, an MLVA (multi-locus variable-number tandem-repeat analysis)-16 assay was performed with 94 B. melitensis isolates. They were identified as B. melitensis biovar (bv.) 1 (67), 3 (10) and Rev. 1 vaccine strains (17) using a classical biotyping and multiplex PCR. In genotyping, three human isolates were grouped at 2 genotypes with sheep isolates, and it implies that B. melitensis are cross-infected between human and livestock. In the parsimony analysis, Mongolian B. melitensis isolates had high genetic similarity with Chinese strains, likely due to the geographical proximity, clustered distinctively as compared with other foreign isolates. B. melitensis Rev. 1 vaccine strains were divided into 4 genotypes with 92% similarity. In the analysis of Rev.1 strains, the risk of mutation of vaccine strain might not be overlooked. Animal quarantines should be strengthened to prevent the spread of Brucella species among adjacent countries.
Subject(s)
Brucella melitensis/genetics , Brucella melitensis/isolation & purification , Brucellosis/epidemiology , Brucellosis/microbiology , Animals , Bacterial Typing Techniques , Brucella/genetics , Brucella/isolation & purification , Brucella Vaccine , Brucella melitensis/classification , Brucella melitensis/immunology , Brucellosis/transmission , Brucellosis/veterinary , China/epidemiology , DNA, Bacterial/genetics , Genotype , Goat Diseases/epidemiology , Goat Diseases/microbiology , Goats , Humans , Molecular Epidemiology , Mongolia/epidemiology , Multilocus Sequence Typing , Multiplex Polymerase Chain Reaction , Phylogeny , Sheep , Sheep Diseases/epidemiology , Sheep Diseases/microbiology , Sheep, Domestic , ZoonosesABSTRACT
To diagnose brucellosis effectively, many genus- and species-specific detection methods based on PCR have been developed. With conventional PCR assays, real-time PCR techniques have been developed as rapid diagnostic tools. Among them, real-time PCR using hybridization probe (hybprobe) has been recommended for bacteria with high DNA homology among species, with which it is possible to make an accurate diagnosis by means of an amplification curve and melting peak analysis. A hybprobe for B. abortus was designed from a specific single-nucleotide polymorphism (SNP) on the fbaA gene. This probe only showed specific amplification of B. abortus from approximately the 14th cycle, given a melting peak at 69°C. The sensitivity of real-time PCR was revealed to be 20 fg/µl by 10-fold DNA dilution, and the detection limit was 4 CFU in clinical samples. This real-time PCR showed greater sensitivity than that of conventional PCR and previous real-time PCR based on Taqman probe. Therefore, this new real-time PCR assay could be helpful for differentiating B. abortus infection with rapidity and accuracy.
