Detection And Molecular Typing Of Milk Brucella Abortus In Cows

In order to investigate the occurrence and epidemic of bovine brucelosis in Zhejiang province, a total of 28243 serum samples were collected from 30 dairy farms in the concentrated area of Jinhua, Zhejiang province, and tested by SAT and RBT for serology survey in 2006. The results showed that 1880 of 28243 serum samples were positive for brucella infection, and the positive rate was 6.66%. Based on the serology survey, 570 of milk samples were collected and used for bacterium isolation, from which 62 strains of bacteria were isolated and propagated using a selective culture medium. The isolated strains were then stained by Gram staining and Kozlowski differential staining, and identified by a serial biochemical tests. The results showed that 50 of 62 strains were consistent with the biochemical indicator characters of reference brucella.A nested-PCR assay for detecting brucella infection in milk. was developed by means of designing outward-directed and inward-directed primers according to brucella's genome sequences, and optimizing the DNA extraction method. The results showed that two rounds of amplification with the two pairs of synthesized 16S rRNA primers can effectively detect the specific DNA fragment of 16S rRNA from the brucella contaminated milk, of which the detection limit was as low as 3.3 CFU/ml. This PCR assay showed high specificity for brucella without any cross-reaction for the related bacteria DNA including Escherichia coli, Hemolytic streptococci, Salmonella, WB bacteria, Staphylococcus aureus, Pseudomonas aeruginosa, and Helicobacter pylori, and was consistent with the bacterium isolation method. Therefore, it is suggested that the nested-PCR assay for brucella with high specificity and sensitivity are very suitable for detecting brucella infection of milk in laboratory.To explore molecular typing for Brucella isolates, we designed specific primers according to DNA polymorphism of omp2 encoding a 31 kDa.outer membrane protein. The omp2 fragments of Brucella isolates were amplified by PCR and sequenced. BLAST in GenBank database showed that 43 of 51 brucella isolates were B. abortus and 8 strains B. millitensis, but their biotypes failed to be identifed. As the method of AMOS-PCR can detect the biotype of the brucella based on the species-specific IS711 gene, which is a transposable element and presents the insert position polymorphism in the difference biotypes of Brucella, the consensus primer and 5 specific primers in downstream matched with B. abortus, B. melitensis, B. ovis, B. suis and DEL569 were then designed for brucella biotyping. The AMOS-PCR identified 35 strains as B. abortus 5, 6, 9 or 3b, 2 strains of B. abortus 1, 2 or 4, and 5 strains of B. melitensis. However, there were 9 strains not be classified into any biotypes. A rpoB gene of B. melitensis with a length of 4134 bp includes all the polymorphism of species and biotype. Therefore, the rpoB gene were cloned and sequenced from the unidentified 9 strains. Three strains of B. melitensis 1, 1 strain of B. abortus1 or 4, and 1 strain of B. abortus 7 were identified, whereas 4 strains of Brucella isolates failed to be identified the biotype of species yet.