• 대한수의학회지
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• 제40권1호
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• pp.86-93
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• 2000

Paratuberculosis (Johne's disease), a chronic enteritis produced by Mycobacterium paratuberculosis, affects a large proportion of ruminants in all continents and causes important economic losses. The identification of well-characterized and species-specific components of M paratuberculosis would provide the means to improve the specificity and sensitivity of immunodiagnostic assays for Johne's disease. The aims of this study were to express the recombinant C-terminal of 34kDa protein (rC34P) of M paratuberculosis in E coli and to investigate the effectiveness of this protein in detecting antibodies to the native protein in sera from paratuberculosis infected cattle. The C-terminal of the gene encoding the 34kDa protein was amplified by polymerase chain reaction from the chromosomal DNA of M paratuberculosis (ATCC 19698) and cloned into vector pGEX-4T-2. Then, cloned plasmid was transformed into E coli DH5${\alpha}$ and the rC34P was overexpressed. The rC34P was purified by affinity chromatography and gel filtration. The rC34P was examined antigenicity by Western blot. The rC34P was reactive with culture positive bovine serum and hyperimmune rabbit anti-M paratuberculosis serum but was not reactive with culture negative bovine serum and tuberculin positive bovine serum in Western blot. In conclusion, the rC34P produced in this study is expected as a useful candidate for antigen in serological diagnosis of Johne's disease.

• 한국임상수의학회지
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• 제27권1호
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• pp.23-28
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• 2010

Mycobacterium avium ssp paratuberculosis, intracellular bacteria that can cause chronic granulomatous enteritis in cattle, continues to pose significant economic losses and health problem with high prevalence. The purpose of this study is the polymerase chain reaction (PCR)-base strategy for early detection of M. avium ssp paratuberculosis in whole blood. Blood samples were collected from korean cattles in Jeonbuk, Korea. The 16 out of 88 serum samples were detected M. partuberculosis by ELISA. Then samples of infected 8 Korean cattles were amplified by PCR. The PCR amplified targets are 16s rDNA and heat shock protein 65kDa (hsp 65). The 16s rDNA provided a highly sensitive and specific tool for the direct detection of mycobacteria. In addition M. avium was confirmed characteristically by the hsp65. Finally there were sure to M. avium ssp paratuberculosis by IS900 PCR. The restriction fragment length polymorphism was identified by PCR amplifications and subsequence restriction enzyme digestions with Pst I of a hsp65. These results indicate that confirm M. avium with 16s rDNA, hsp65 and a restriction fragment length polymorphism in the hsp65 gene can be seem the other pattern. Therefore, these results can be used for clinical direct detections of M. avium ssp paratuberculosis in whole blood of Korean cattle and also to be used epidemiological researches.

• 대한수의학회지
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• 제24권1호
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• pp.58-63
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• 1984

Fecal material from cattle, which was confirmed to be infected with Johne's disease by clinical and pathological symptoms, was decontaminated with 4% NaOH and inoculated into the $L{\ddot{o}}wenstein$-Jensen media supplemented with 1% of heat-killed Mycobacterium bovis. After 2-4 week-incubation at $37^{\circ}C$, typical acid-fast mycobacteria was isolated. With the results of staining properties, morphological characteristics, the requirement of mycobactin for growth and the other biochemical properties, isolated mycobacteria was identified as Mycobacterium paratuberculosis. Female guinea pigs were sensitized with the isolates, and skin test was done with purified protein derivatives (PPDs) of M. avium, M. bovis and M. paratuberculosis 4 weeks after sensitization. Animals showed the largest reaction to the PPDs of M. avium and M. paratuverculosis.

• 대한수의학회지
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• 제42권1호
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• pp.81-88
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• 2002

The purpose of this study was to conduct diagnosis of bovine paratuberculosis in Kangwon area. Blood samples were collected from 2,261 dairy cows of 162 herds, and the ELISA and immunoblotting using recombinant 34KDa protein of M. paratuberculosis were conducted. The feces collected from dairy cows were cultured on HEY medium with mycobactin-J and PCR was conducted with washing solution of medium 4 weeks after culture. The ELISA had sensitivity of 83.3% and specificity of 96.7%. And the immunoblotting had sensitivity of 83.3% and specificity of 100%. Of the 2,261 dairy cows, 371 cows(16.4%) were positive in ELISA and 75 cows(3.3%) were positive in immunoblotting. And of the 162 herds, 109 herds(67.3%) had an apparent paratuberculosis prevalence by ELISA and 40 herds(24.7%) by immunoblotting. The geographic distribution of herds with paratuberculosis was not uniform. Of the 241 feces samples including 110 feces from ELISA positive cow, 9 feces were positive in culture and PCR. PCR was able to detect the growth of M. paratuberculosis as early as 4 weeks of culture.

• 대한수의학회지
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• 제54권1호
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• pp.55-57
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• 2014

Paratuberculosis caused by Mycobacterium avium subsp. paratuberculosis (MAP) has extended latent periods of infection. Due to this property, difficulties in the detection of fecal shedder have been raised. A newly designed method for DNA extraction from fecal specimens, mGITC/SC was evaluated in terms of diagnostic efficiency. The detection limit of IS900 real-time PCR was about 50 MAP (1.5 cfu) in 250 mg of feces (6 cfu per g). Also, this DNA extraction method was faster and cheaper than that using commercial kit or other methods. Consequently, the mGITC/SC is an economical DNA extraction method that could be a useful tool for detecting MAP from fecal specimens.

• 대한수의학회지
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• 제37권2호
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• pp.349-358
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• 1997

Mycobacterium paratuberculosis is the etiologic agent of Johne's disease, a chronic inflammatory bowel syndrome in ruminants. The attempts to control or eradicate the disease were severely hampered by the inadequacies of present diagnostic methods. The first purpose of this study was to detect Johne's disease out of 577 cows in the province of Kyunggi, Chungchong, Gangweon and the second purpose was to compare the results of non-absorbed ELISA, absorbed ELISA, PCR, and conventional culture methods. The third purpose was to increase diagnostic specificity, accuracy and rapidity. When non-absorbed ELISA test was conducted with Mycobacterium paratuberculosis antigen, the prevalence of positive was 10.9%. To increase diagnostic specificity, absorbed ELISA test with Mycobacterium phlei was used. In this test, the positive prevalence was 1.7%. For the specific detection of Mycobacterium paratuberculosis, PCR was applied to bacterial culture obtained from fecal samples of cattle. The DNA sequences derived from IS900 were used to prepare DNA primers for detection and identification of Mycobacterium paratuberculosis by PCR. PCR for M paratuberculosis isolated from fecal cultures amplified specific target DNA. PCR was much more rapid than that obtained by conventional culture technique in diagnosis of Johne's disease.

• 한국동물위생학회지
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• 제30권1호
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• pp.95-102
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• 2007

The purpose of this study was to investigate the seroprevalence of infection with the production-limiting diseases in dairy cattle in Jeonbuk-Iksan area. The blood samples were collected from 260 dairy cows in 52 herds, and examined. The antibody rates against N caninum, M paratuberculosis, and bovine leukemia virus were 34.6%, 13.5% and 89.6%, respectively. All samples for bovine brucellosis were negative.

• 한국동물위생학회지
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• 제32권3호
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• pp.251-255
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• 2009

TThis survey was carried out to investigate the prevalence of the antibody for bovine paratuberculosis (Johne's disease) in dairy cattle in Seosan-Taean area. From February to August in 2009, 254 M.RT. samples were collected from 57 farms in the regions and enzyme immuno-sorbent assay (ELISA) was conducted. Among 254 samples, 13 (5.1%) M.R.T. samples of 3 (5.2%) farms were positive by ELISA. In regional analysis, 1 (3.1%) of 34 farms in Seasan and 2 (8.6%) of 23 farms in Taean were positive in ELISA. According to the raising scale of dairy farms, the farm with below 30 heads showed the higher positive rate (2 out of 3 positive farms) than the farms with over 30 heads (1 out of 3 positive farms).

• 대한의생명과학회지
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• 제6권1호
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• pp.65-72
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• 2000

반추수에서 발생하는 Johne병의 조기 진단 방법을 제시하고 이 질병의 원인체와 미생물학적 특징 이 유사한 M. bovis, M. avium 등의 mycobacteria 감염증을 감별 진단하는 방법 을 개발하기 위하여 Mycobacterium 균속의 표준균주를 사용하여 중합효소 연쇄반응을 확립하였다. Johne병으로 의심되는 소의 혈액과 유즙을 채취하여 분리한 단핵구 및 거식세포로부터 genomic DNA를 추출하였다. 각 시료로부터 추출한 DNA를 template로 이용하여 Mycobacterium spp.에 특이적인 16S rDNA primer set를 이용한 PCR을 수행하여 시료내의 mycobacterial DNA 보유 여부를 확인하였다. 한편 mycobacteria 양성으로 확인된 시료는 M. avium complex 균종에 특이한 16S rDNA 염기서열을 기초로 하여 제작한 primer set와 M. paratuberculosis의 IS900 sequence에 특이한 primer set를 이용하여 duplex PCR을 수행하여 Johne병 원인체의 보균 여부를 조사하였으며, 이 결과를 oligonucleotide probe를 사용한 Southern blot hybridization을 통하여 다시 확인하였다. 이와 같은 duplex PCR기법을 실제 축산 현장에서 수집한 유즙과 말초혈액으로부터 분리한 단핵구 및 거식세포 시료에 적용한 결과 본 연구에서 확립한 duplex PCR기법 유용성을 확인할 수 있었다.

• BMB Reports
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• 제47권2호
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• pp.115-120
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• 2014

In this study, we show that Mycobacterium avium subsp. paratuberculosis MAP1305 induces the maturation of bone marrow-derived dendritic cells (BMDCs), a representative antigen presenting cell (APC). MAP1305 protein induces DC maturation and the production of pro-inflammatory cytokines (Interleukin (IL)-6), tumor necrosis factor (TNF)-${\alpha}$, and IL-$1{\beta}$) through Toll like receptor-4 (TLR-4) signaling by directly binding with TLR4. MAP1305 activates the phosphorylation of MAPKs, such as ERK, p38MAPK, and JNK, which is essential for DC maturation. Furthermore, MAP1305-treated DCs transform naive T cells to polarized $CD4^+$ and $CD8^+$ T cells, thus indicating a key role for this protein in the Th1 polarization of the resulting immune response. Taken together, M. avium subsp. paratuberculosis MAP1305 is important for the regulation of innate immune response through DC-mediated proliferation of $CD4^+$ and $CD8^+$ T cells.