• 대한수의학회지
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• 제45권2호
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• pp.199-205
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• 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.

• 대한수의학회지
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• 제55권2호
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• pp.105-110
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• 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.

• 한국동물위생학회지
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• 제30권2호
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• pp.251-258
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• 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.

• 한국동물위생학회지
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• 제28권4호
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• pp.387-392
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• 2005

This study was attempted to investigate the properties of brucellosis in Korean indigenous cattle at the Yeongwol and Pyeongchang county. Brucella spp was differentiated and identified from cotyledons, amniotic fluids and supramammary lymph nodes which confirmed with clinical, serological, epidemiological evidences (69 cases) from January to June, 2004. Isolation frequency of this causative agent from supramammary lymph nodes, cotyledons and amniotic fluids from 38 pregnant Korean indigenous cattle were $39.1\%,\;87.5\%,\;and\;63.2\%$, respectively, and finally confirmed with Brucella abortus biotype 1 through biochemical and serological test. A Brucella specific DNA with 711bp band was detected by PCR assay using BCSP primer. The two cases were definite epidemiological evidences that infected Korean indigenous cattle acrossed the border to Yeongwol and Pyeongchang from near two provinces. Effective prevention programs are urgently needed for further spreading this epidemics.

• 한국동물위생학회지
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• 제27권2호
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• pp.159-164
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• 2004

A dairy farm that has been suffered continuously(more than 2 years) from brucellosis in Korea in spite of repeated legal test-and-slaughter was investigated the main source of infection in the farm. All cattle(22 milking cows, 44 heifers, 60 calves, 8 bull), dogs(3 mixed breed), feces from wild birds(3 samples), drinking water(3 sites), and soil in the paddocks(14 sites) inside the farm were examined with serological and/or bacteriological methods including specific DNA detection with PCR method. Brucella spp in the milk and blood were detected in 12/22 and 5/22 milking cows, respectively, although all of them were negative with conventional tube agglutination test. The number of serologically positive heifer was 15(15/44), but the isolation of Brucella spp was succeeded in the only 11(11/15) of them. Brucella were detected in vagina 1(1/11) and nasal(3/12) excretion in serologically positive heifers. All the three dogs were serologically positive, and Brucella spp were isolated from their blood. However, Brucella spp were not detected in the drinking water, soil in the paddocks, nor the feces of wild birds. The results suggest that milking cow secrete Brucella spp through milk, genital tract and nasal cavity, which are the major source of infection in this farm, The main infection route of Brucella spp is contact to contact with Brucella spp excreting animals rather than environmental contamination. The animals, living together with infected cow such as dogs, are the readily susceptible and are required to be examined for Brucella spp.

• 대한수의학회지
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• 제38권2호
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• pp.345-352
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• 1998

The diagnosis of brucellosis is currently based on serological and microbiological tests. However, the microbiological isolation and identification have several disadvantages such as time-consuming and laborious, and the serological methods have been reported to cross-react with antigens other than those from Brucella spp. To develop a sensitive and rapid diagnostic method for detection of Brucella species, the genus-specific primers were designed and synthesized from the sequence of gene encoding a 31kDa cell surface protein(BCSP) and a 36kDa outer membrane protein(OMPB) of B abortus. The amplified 711bp and 982bp DNA fragments were only visible in each species of Brucella by PCR method using the BCSP and OMPB primers, respectively. However, PCR product was not obtained with DNA from other Gram-negative bacteria. As little as 1pg of the B abortus genomic DNA could be detected by this PCR method. Using the PCR technique, semen samples from 185 bulls of Brucella-seronegative herds in Cheju island were examined for comparison of this PCR method with conventional methods in 1995. The semen samples from 5 bulls were positive by culture method and PCR, and one was positive and 5 were suspect by semen plasma agglutination test. However, the semen samples obtained from 177 bulls were negative by semen plasma agglutination, culture and PCR methods in 1996. The results of comparison tests suggested that PCR was a better test than agglutination test against semen of bulls. This study indicated that the PCR technique was a valuable for the diagnosis of bovine brucellosis, particulary in bull semens.

• 한국동물위생학회지
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• 제30권3호
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• pp.375-384
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• 2007

This study was conducted to investigate the characteristics of brucellosis in Korean native cattle in a farm where bovine brucellosis was confirmed 3 times from September 2006 to March 2007. Of 74 bulls serum samples examined, 21 (28.4%) were positive by Rose-Bengal test (RBT) and Standard tube agglutination test (STAT). In the isolation test from seropositive bulls, B abortus was isolated and identified from 2 specimens (testis, intestinal lymph node) among 6 kinds of specimens including blood, urine, feces and soil. Isolation rate of intestinal lymph node and testis was 25% (3/12 cases) and 16.7% (2/12), respectively. B abortus was also isolated from calves below 12 months old, i.e., 1 isolate (25.0%) was confirmed from testis, 4 (40.0%) from supra-mammary lymph nodes and 1 (25.0%) from intestinal lymph node. All isolates had Brucella specific 16s r-RNA with 905-bp band detected by PCR assay. For the more effective control of bovine brucellosis in korea, this paper would like to suggest that all of bulls and calves should be included in the screening tests.

• Journal of Preventive Veterinary Medicine
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• 제43권4호
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• pp.214-220
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• 2019

Although canine brucellosis has been known to be an important re-emerging zoonosis, the pathophysiological mechanisms of Brucella canis infection remains clues to be solved. Different culture models, single and co-culture models, were constructed with canine epithelial cells, D17 and macrophage, DH82 to investigate the induction of immune responses in in vivo B. canis infection. Expression of genes related with induction of immune responses, Th1, Th2 and Th17, was compared in the two different models after the bacterial infection. In this study, expression of cytokine genes, IL-1β, IL-5, IL-6, IL-10, IL-23, and TNF-α was quantified in the DH82 at different time points using RT-qPCR in the two different culture systems after the infection. Cytokine genes related with Th1, IL-1β and TNF-α and Th17, IL-6 and IL-23 were expressed with time-dependent manners in the both systems (p<0.05). However, increase of Th2-related cytokine genes expression was not detectable in the both systems by comparison with control. The expression of Th1 and Th17 related cytokine genes was earlier in single cell culture than those in co-culture model (p<0.05). In general, amounts of the expressed genes were shown higher in single cell model than those in co-culture models. This study indicate that Th1 and Th17-associated immune responses are central to B. canis infection in dogs. In addition, it suggests a specific role of epithelial cells in the B. canis infection in vivo, which should resolved in the further study.

• 대한수의학회지
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• 제40권3호
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• pp.533-541
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• 2000

Biochemical and genetic analysis were carried out to investigate the potential recovery of pathogenecity or related mutations of Brucella abortus RB51 vaccine strains. RB51 strains were recovered from commercial vaccines, including related seed stocks from private companies in Republic of Korea, strain from USA, a reference strain from C university and a field isolate (Daehungjin) from aborted dairy cow after RB51 vaccination were compared with two identified virulent wild strains (S2308 and a field strain isolated from dairy cow in Korea) at the same conditions. All the strains examined, except identified pathogenic strains, revealed the identical characteristics to the original RB51 in biochemical properties, antigen and bacteriophage typing. Outer membrane protein (OMP) profiles from strains of RB51 showed the same patterns with standard RB51 in SDS-PAGE. In addition, Western blotting with the brucella specific monoclonal antibody also indicated that all the vaccine strains were completely deficient in their LPS compared to the pathogenic Br abortus strains. The differences in DNA structures among strains were also possible to detect after PCR. All vaccine strains, except S19, S1119-3, S1075, S544 and Br suis, were amplified a 178bp DNA fragment of eri-gene, and 364bp of IS711 elements. In contrast, 498bp DNA product was only found with Br abortus. Overall evidences in the present study confirmed that the RB51 strains for vaccine production in Korea did not originated from the phenomena of possible recovery of pathogenicity or related to any potential mutation event at all.

• 한국동물위생학회지
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• 제33권2호
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• pp.135-141
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• 2010

Bovine brucellosis, an important zoonosis, is diagnosed with serological tests such as the RBT, TAT using inactivated whole bacterial cells or bacterial lipopolysaccharide (LPS) antigen in Korea. However, a strong cross-reaction between Brucella spp. and Yersinia enterocolitica O9 in these tests has seriously complicated the diagnosis of animal brucellosis because Brucella spp. shares common antigenic determinants with Y. enterocolitica O9 in the smooth LPS region. In this study, Brucella-field strains were isolated from Brucellapositive Hanwoo in Kimhae, Korea and outer membrane protein (omp) which has low cross-reaction with Y. enterocolitica O9 and high immunogenicity was extracted from the field strains Then we compared ELISA using the extract with RBT-TAT. Fifteen field strains were isolated from 47 supra-mammary-lymph nodes, which were collected from 18 farms. Isolation rate was 32%. Brucella-specific antigen was identified by performing SDS-PAGE or Western blotting on extracted omp with at 0.5% n-lauroylsarcosine One hundred and ninety-two serum-samples were used in the experiment: 142 negative and 50 positive samples verified by RBT-TAT. According to ELISA results, 127 samples were negative and 15 appeared positive among 142 negatives by RBT-TAT, while 42 samples were positive and 8 were negative among 50 positives by RBT-TAT. Therefore, it showed 89.4% of specificity and 84% of sensi-tivity. Through the current experiments, we could set up an ELISA based on the omp which has low cross-reaction and high immunogenicity and concluded that the omp could be a good material for accurate diagnosis of bovine brucellosis.