• Korean Journal of Veterinary Research
• /
• v.45 no.2
• /
• pp.199-205
• /
• 2005

DNA fingerprint patterns of 8 Brucella reference strains and 15 B. abortus field isolates were characterized by repetitive element sequence-based PCR (rep-PCR) using BOX- and ERIC-primers in this study. AMOS PCR differentiated all Brucella field isolates from B. abortus RB51, a vaccine strain by producing a B. abortus-specific 498 bp band. Rep-PCR using BOX-primer produced 13 to 18 bands with sizes of between 230 and 3,300 bp, and discriminated Brucella strains to the species level except B. canis and B. suis. PCR products amplified with ERIC primers were, however, not appropriate for differentiating the Brucella isolates. DNA fingerprint patterns for all B. abortus field isolates were identical among them and were put on one cluster with B. abortus biovar 1 reference strain in the dendrogram, indicating they were highly clonal. These results suggested that rep-PCR using BOX primer might to be a useful tool for calculating genetic relatedness among the Brucella species and for the study of brucellosis epidemiology.

• Korean Journal of Veterinary Research
• /
• v.52 no.2
• /
• pp.75-82
• /
• 2012

We have developed and optimized a multiplex polymerase chain reaction (mPCR) for simultaneous detection of Brucella, Salmonella and Leptospira with high sensitivity and specificity. Three pairs of oligonucleotide primers were designed to specifically amplify the targeted genes of Salmonella, Leptospira and Brucella species with sizes of 521, 408 and 223 bp, respectively. The mPCR did not produce any nonspecific amplification products when tested against 15 related species of bacteria. The sensitivity of the mPCR was 100 fg for Brucella and 1 pg for both Salmonella and Leptospira species. In the field application, kidney, liver and spleen were collected from wild rats and stray cats and examined by mPCR. The high specificity and sensitivity of this mPCR assay provide a valuable tool for diagnosis and for the simultaneous and rapid detection of three zoonotic bacteria that cause disease in both humans and animals. Therefore, this assay could be a useful alternative to the conventional method of culture and single PCR for the detection of each pathogen.

• Korean Journal of Veterinary Service
• /
• v.35 no.4
• /
• pp.275-281
• /
• 2012

Canine brucellosis is the zoonosis in worldwide and Brucella (B.) canis is a facultative intracellular pathogen that has a very limited host. MLVA-16 (Multilocus VNTR analysis) is a efficient method for genotyping of Brucella species. Various methods have been established for genotyping of Brucella species, but most of analytical method is lack reproducibility and limited capability to differentiate them. B. canis isolates (n=73) from 7 farms in Gyeongbuk province in 2003~2010 were analyzed using 16 VNTR loci. Automatic electrophoresis system was utilized for more high throughput and rapid simple discrimination. Thirty two genotypes were identified from 73 B. canis isolates. MLVA could contribute to molecular typing for epidemiological evaluation of canine brucellosis.

• Korean Journal of Veterinary Service
• /
• v.34 no.3
• /
• pp.227-234
• /
• 2011

Brucella (B.) abortus is a facultative intracellular pathogen that infects a wide variety of animal species and human. Brucellosis is the zoonosis and an extremely important disease around the world. Although the eight species can be differentiated by conventional phenotypic tests, these species display a high degree of DNA homology in DNA-DNA hybridization assay (>90%). Various methods have been established for genotyping of Brucella species, but most of analytical methods are lack reproducibility and limited capability to differentiate them. The attempt of this study was to evaluate multiple-locus VNTR analysis (MLVA) for use of epidemiological trace-back analysis in bovine brucellosis. Ninety-four B. abortus isolates from Gyeongbuk province during 2006~2010 were analyzed using 16 VNTR locus. High resolution automatic capillary electrophoresis system was used for more throughput, simpleer, faster, and better discriminable than conventional gel electrophoresis. As a result, 13 different genotypes were identified from 94 B. abortus isolates. MLVA could contribute to epidemiological trace-back analysis of bovine brucellosis.

• Korean Journal of Veterinary Research
• /
• v.55 no.2
• /
• pp.105-110
• /
• 2015

A real-time PCR assay using hybridization probe (HybProbe) has been developed to detect Brucella (B.) melitensis strains. The primer and HybProbe sets were designed based on the gap gene of chromosome I with a specific single nucleotide polymorphism of B. melitensis. Specificity of the assay was confirmed by comparison to reference Brucella species and other related strains. In the melting curve analysis, B. melitensis generated a peak at $67^{\circ}C$ unlike those for other Brucella species observed at $61^{\circ}C$. Sensitivity of the assay for B. melitensis ranged from 20 ng to 200 fg of genomic DNA. The ability to identify 94 Mongolian B. melitensis isolates using the real-time PCR assay was identical to that of classical biotyping methods and differential multiplex PCR. These data showed that this new molecular technique is a simple and quick method for detecting B. melitensis, which will be important for the control and prevention of brucellosis.

• Korean Journal of Veterinary Service
• /
• v.30 no.2
• /
• pp.251-258
• /
• 2007

Bovine brucellosis has occurred for years in Gyeonggi province under the national test and slaughter scheme. The serum agglutination test (SAT) is a diagnostic tool to confirm the disease despite the argument on its specificity. We selected 8 farms where only one or two individuals were diagnosed as brucellosis through SAT at the primary regular herd check and isolated the causative organism and characterized the species by species-specific PCR. The pathogen isolation was successful in 6 farms out of 8 farms by microbiological culture, showing the successful rate of 75%. The isolation rate of the causative organism represents 70% from supra-mammary lymph node and 60% from uterine tissues. They were characterized as Brucella abortus biovar 1 after biotyping by PCR, showing the fragment of 498 bp. Five of 8 farms were diagnosed as brucellosis two to four times more over the intervals of two or three months. Here in this study we briefly showed the correlation of the sporadic outbreak of brucellosis tested by SAT and the isolation of the causative organism. Moreover one or two reactors against brucellosis among considerable size of herd may indicate that SAT failed to detect potentially infected individuals in the incubation stage or chronic phase of the disease.

• Korean Journal of Veterinary Research
• /
• v.56 no.3
• /
• pp.147-153
• /
• 2016

A total of 782 blood and 465 tissue samples from 1,039 wild animals and 127 dairy goats were collected from January 2011 to December 2013 in 10 provinces of South Korea and tested for the presence of brucellosis. The Rose Bengal test revealed that 8.0% (52/650) of the serum samples were seropositive, while 4.2% (33/782) of the serum samples were positive for Brucella antibodies by competitive enzyme-linked immunosorbent assay. Of the 650 sera examined, only 16 (2.5%) were positive by both serological tests. Direct polymerase chain reaction (PCR) assay using B4/B5 primers for Brucella abortus (BCSP31) revealed the prevalence of Brucella to be 26.5% (129/487) in blood samples and 21% (98/465) in tissue samples while, 16S rRNA PCR detected Brucella DNA in 6.8% (33/487) and 2.6% (12/465) in blood and tissue samples, respectively. Of PCR-positive samples, only 6.2% (30/487) of blood samples and 2.4% (11/465) of tissue samples were found to be positive by both BCSP31 and 16S rRNA PCRs. However, Brucella strains were isolated by blood culture from only two out of 487 blood samples (0.4%). This characterization and identification of pathogenic Brucella isolates is the first to clearly indicate that the organisms were Brucella abortus biovar 1.

• Journal of Microbiology and Biotechnology
• /
• v.33 no.4
• /
• pp.441-448
• /
• 2023

Brucellosis is a contagious zoonotic disease that infects millions of people annually with hundreds of millions more being exposed. It is caused by Brucella, a highly infectious bacterial species capable of infecting humans with an estimated dose of 10-100 organisms. Sirtuin 1 (SIRT1) has been reported to contribute to prevention of viral diseases as well as a chronic infection caused by Mycobacterium bovis. Here, we investigated the role of SIRT1 in the establishment of Brucella abortus infection in both in vitro and in vivo systems using the reported SIRT1 activators resveratrol (RES), piceatannol (PIC), and ginsenoside Rg3 (Rg3). In RAW264.7 cells, SIRT1 activators did not alter the adherence of Brucella or Salmonella Typhimurium. However, reduced uptake of Brucella was observed in cells treated with PIC and Rg3, and survival of Brucella within the cells was only observed to decrease in cells that were treated with Rg3, while PIC treatment reduced the intracellular survival of Salmonella. SIRT1 treatment in mice via oral route resulted in augmented Brucella resistance for PIC and Rg3, but not RES. PIC treatment favors Th2 immune response despite reduced serum pro-inflammatory cytokine production, while Rg3-treated mice displayed high IL-12 and IFN-γ serum production. Overall, our findings encourage further investigation into the complete mechanisms of action of the different SIRT1 activators used as well as their potential benefit as an effective alternative approach against intracellular and extracellular pathogens.

• Journal of Microbiology and Biotechnology
• /
• v.21 no.1
• /
• pp.89-92
• /
• 2011

The routine identification and differentiation of Brucella species is a time-consuming and labor-intensive process, which frequently places personnel at risk of laboratory-acquired infection. Here, we describe the development of a rapid multiplex PCR assay for the confirmation of presumptive Brucella isolates. The assay was able to identify and differentiate major human pathogens, namely B. abortus, B. melitensis, and B. suis, in a single test of less than an hour and a half.

• Korean Journal of Microbiology
• /
• v.32 no.1
• /
• pp.7-11
• /
• 1994

The free-livein nitrogen fixing bacterium, Arcobacter nitrofigilis which has been known to be a microaerophile, exhibited aerobic growth in brucella broth. In a level of oxygen equivalent to an air atmosphere (21% $O_2$), the maximum cell growth was observed in brucella broth. Low level of cell growth occurred in a level of low oxygen equivalent to lower than 2%, unless any other terminal electron acceptors other than oxygen were wupplied in brucella broth. Membrane-bound cytochrome b and c, and soluble cytochrome c were found. The growth in an aerobic atmosphere, little growth at low oxygen level, and occurrence of cytochrome c mean that this species is an aerobe and obtains energy using energy-yielding respiration.