Epidemiological Investigation Of Brucellosis In Livestock And Molecule Typing Of Brucella Based On Single Nucleotide Polymorphism (SNP) Analysis

Brucella is the pathogenic bacterium of Brucellosis, which can infect numerous livestock, such as bovine, ovine, swine, canine, wildlife animals and human. As a reemerging zoonotic, Brucellosis has a worldwide discribution, which leads to humorous economic loss and serious problem of public health. Since the late 90s of last century, brucellosis in China increases year by year, so a comprehensive epidemiological investigation of brucellosis was extremely necessary. The genus Brucella was divied into several species and biovars, which were different in host preference, biological traits, virulence strength, clinical features, epidemiological characteristics. So rapid diagnosis was important for epidemiological tracing and development of the project to prevent and control Brucellosis. Although as the "gold standard", traditional biotyping of Brucella was time-consuming, subjective and lacking of accuracy, whatmore the method had the potential risk of infection. BSCP31-PCR, AMOS-PCR and other methods also had some limitations. So there was an urgent need to establish a new, rapid, easy and accurate diagnosis of Brucella.To explore and develop a new tool for the rapid, simple and unambiguous characterization of Brucella, several housekeeping gene and other conserved gene sequences where analyzed to screen single nucleotide polymorphism (SNP). According to allele discrimination, Real-time PCR was construceted to Brucalla genus, species and biovar, and then applied in epidemiological investigation of Brucellosis comparing with other biotyping and PCR aprroch. The main experiments and results are as follows:1 Construction of Real-Time PCR Assays based on Single-Nucleotide Polymorphisms for rapid indentification of Brucella isolates.This article describes new single nucleotide polymorphism (SNP) signatures for the rapid identification of Brucella, species characterization of the six classical Brucella spp and the mammal Brucella, and biotyping of B.melitensis all biovars and B.abortus partial biovars. Fourteen pairs of short sequence Minor Groove Binding (MGB) TaqMan probes were designed corresponding to SNP based on alignment and analysis of several housekeeping gene and other conservaed gene sequences.The scope of the assay was confirmed by testing all Brucella reference strains (except B.suis biovar 5), vaccine strain and close phylogenetic relatives such O.anthropi. The assay is specific to positive discrimination of Brucella from its closest phylogenetic neighbours, sensitive being capable of detecting and differentiating down to 50fg (15 genome equivalents). Furthermore, this article constructed the positive control for both qualitative and quantitative analysis in around two hours. Clone the target gene with the specific SNP loci for building the positive control, so rapid detection and typing of Brucella not only in the qualitative but also in quantitative. These results suggest that the molecular typing method for the genus Brucella, subspecies and some subtypes was established successfully.2 Epidemiological investigation of livestock Brucellosis.In order to find out the prevalence of livestock Brucellosis,12989 unvaccined bovine sera,11303unvaccined ovine sera were collected from 14 provinces or area,27473 unvaccined suis sera were collected from 7 provinces or area, and 5119 unvaccined yalk sera from 5 provinces or area in western China. All sera were detected with RBPT and SAT, and positive were confirmed with I-ELISA and C-ELISA, recommended by OIE. The positive rate of ovine sera was 7.01%, compared bovine sera with 6.00%. Bovine Brucellosis in Nothen-East China and ovine Bruellosis in Nothern China were the most seriously. Whatmore, the positive rate of animal feeding in large scale were higher than animal feeding alone. Furthermore, there was outbreak of Brucellosis and local prevalence in spatial area of China. The Brucellosis of yalk was mucn serious than expectation with the positive rate of 3.69%, and the infected area was wider than expectation too. During 2008 to 2010,45 field strains were isolated from aborted fetus, milk and blood. Of 45 field isolates,14 were B.melitensis,8 were B.suis,3 were B.canis, and the others were B.abortus. Field isolates wre diagnosed and discriminated with biotyping, PCR and Real-time PCR, and the coincidence rate was 100%. The assay described in this article represents a new tool for the rapid, simple and unambiguous characterization of Brucella to the species and biovar level, and will be widely applicable in future.