• Proceedings of the Korean Society of Veterinary Service Conference
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• 1997.05a
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• pp.240.1-240
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• 1997

• Korean Journal of Veterinary Research
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• v.41 no.4
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• pp.535-542
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• 2001

A multiplex PCR technique was developed for detecting specifically each Mycobacterium bovis, M. tuberculosis, M. avium and M. avium subsp, paratuberculosis, respectively, using clinical samples of field cattle. To apply this novel technique to clinical specimens, blood sample was obtained from live cows comprising 11 intradermal tuberculin test (ITT)-positive and 17 ITT-negative and tested by multiplex PCR. Positive results were obtained from 15 cows by the multiplex PCR, showing that 4 (23.5%) of the 17 ITT-negative cows were multiplex PCR positive. The multiplex PCR results also showed that among the 15 positive cows, 7 (46.7%) were infected with M. bovis, 1 (6.7%) with M. tuberculosis and 7 (46.7%) with M. avium. The sensitivity and specificity of multiplex PCR in comparison with those of ITT were 100% and 76.5%. The correlation between the multiplex PCR and ITT assays with blood samples was considered excellent, 85.7% agreement and ${\kappa}=0.72$. The results obtained, using reference mycobacterial strains and typed clinical samples, show that the multiplex PCR method may be a rapid, sensitive, and specific tool for the differential identification of various mycobacterial strains in a single-step assay. Therefore, multiplex PCR assay is a useful tool for early diagnosis of tuberculosis in live cattle and to identify the species or complex of mycobacterium from clinical samples.

• Journal of Life Science
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• v.15 no.3 s.70
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• pp.415-422
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• 2005

library of the mutants was prepared by transposon mutagenesis of the Mycobacterium bovis BCG. We screened this library for the resistance to an anti-tuberculosis antibiotic, PA-824. Most of the mutants resistant to the PA-824 were not able to synthesize the coenzyme $F_{420}$ which is normally produced by the wild type M. bovis BCG strains. HPLC analysis of the cellular extract showed that one of those mutants which lost the ability to synthesize $F_{420}$ still produced F0. The insertion site of the transposon in this mutant was determined by an inverse PCR and the transposon was found to be inserted in the Rv2435c open reading frame (ORF). Rv2435c ORF is predicted to encode an 80.3 kDa protein. Rv2435c protein appears to be bound to the cytoplasmic membrane, its N-terminal present in the periplasm and C-terminal in the cytoplasm. The C-terminal portion of this protein is highly homologous with the adenylyl cyclases of both prokaryotes and eukaryotes. There are 15 ORFs which have homology with the class III AC proteins in the genome of the M. tuberculosis and M. bovis. Two of those, Rv1625c and Rv2435c, are highly homologous with the mammalian ACs. We cloned the cytoplasmic domain of the Rv2435c ORF and expressed it with six histidine residues attached on its C-terminal in Escherichia coli to find out if this protein is a genuine AC. Production of that protein in E. coli was proved by purifying the histidine-tagged protein by using the Ni-NTA resin. This protein, however, failed to complement the cya mutation in E. coli, indicating that this protein lacks the AC activity. All of the further attempts to convert this protein to a functional AC by a mutagenesis with UV or hydroxylamine, or construction of several different fusion proteins with Rv1625c failed. It is, therefore, possible that Rv2435c protein might affect the conversion of F0 to $F_{420}$ not by synthesizing cAMP but by some other way.

• BMB Reports
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• v.34 no.4
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• pp.342-346
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• 2001

The Mycobacterium bovis BCG panB gene, encoding ketopantoate hydroxymethyltransferase (KPHMT), was cloned from a ${\lambda}gt11$ genomic library and sequenced. The DNA sequence encodes a protein that contains 281 amino acid residues (M, 29,337) with a high similarity to the KPHMTs. Subcloning of a 846 by open reading frame (ORF), but not a 735 by ORF, into the vector pUC19 led to complementation of the panB mutant of Escherichia coli. The BCG pang gene was overexpressed in E. coli and the KPHMT purified to homogeneity The recombinant protein was further confirmed by an enzymatic assay.

• Korean Journal of Veterinary Research
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• v.24 no.1
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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.

• Tuberculosis and Respiratory Diseases
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• v.72 no.6
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• pp.475-480
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• 2012

Background: Pyrazinamide (PZA) is an effective antitubercular drug that becomes toxic to Mycobacterium tuberculosis when converted to pyrazinoic acid by pyrazinamidase (PZase), encoded by mycobacterial pncA. A strong association was noted between the loss of PZase activity and PZA resistance. The causative organisms in extrapulmonary tuberculosis are rarely cultured and isolated. To detect pncA mutations in specimens from extrapulmonary tuberculosis as confirmative diagnosis of mycobacterial infection and alternative susceptibility test to PZA. Methods: Specimens were collected from clinically proven extrapulmonary tuberculosis. pncA was sequenced and compared with wild-type pncA. Results: pncA from 30 specimens from 23 donors were successfully amplified (56.6% in specimens, 59% in donors). Six mutations in pncA were detected (20.0% in amplified specimens, 26.1% in specimen donors) at nucleotide positions of 169, 248 and 419. The mutation at position 169 results in substitution of aspartic acid for histidine, a possible allelic variation of M. bovis that have intrinsic PZA resistance. The mutation at position 248 changes proline into arginine and that at position 419, arginine into histidine. Conclusion: DNA-based diagnosis using pncA may be simultaneously useful for the early diagnosis of mycobacterial infection and the rapid susceptibility to PZA in extrapulmonary tuberculosis. A potential implication of pncA allelic variation at 169 might be suggested as a rapid diagnostic test for M. bovis infection or Bacille Calmette-Gu$\acute{e}$rin (BCG) reactivation.

• The Journal of the Korean Society for Microbiology
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• v.35 no.2
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• pp.149-157
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• 2000

Mycobacterium tuberculosis is capable of growing and survival within macrophage. The purpose of this study was to identify the genes regulated by infection of mycobacteria in human monocytic THP-1 cells. We used the differential display reverse transcriptase polymerase chain reaction (DD RT-PCR) and nothern blot analysis to confirm the differentially expressed genes from THP-1 cells infected with live Mycobacterium tuberculosis H37Rv, heat-killed Mycobacterium tuberculosis H37Rv and live Mycobacterium bovis BCG. Among many up or down-regulated clones, 27 clones were sequenced and compared with known genes on GenBank. Thirteen of over-expressed clones from THP-1 cells infected with live Mycobacterium tuberculosis H37Rv were identical to human prothymosin alpha, eight were novel clones and six clones showed homology with Human ferritin H chain, Esherichia coli bgl, Mouse RNA-dependent EIF-2 alpha kinase, E. coli htrL, Hyaluronan receptor and T cell receptor. Our result suggests that Mycobacterium tuberculosis might regulate prothymosin alpha gene transcription in monocytic THP-1 cell.

• Journal of Microbiology
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• v.45 no.3
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• pp.268-271
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• 2007

In Korea, the Mycobacterium tuberculosis K-strain is the most prevalent clinical isolates and belongs to the Beijing family. In this study, we conducted comparative porteomics of expressed proteins of clinical isolates of the K-strain with H37Rv, H37Ra as well as the vaccine strain of Mycobacterium bovis BCG following phagocytosis by the human monocytic cell line U-937. Proteins were analyzed by 2-D PAGE and MALDI-TOF-MS. Two proteins, Mb1363 (probable glycogen phosphorylase GlgP) and MT2656 (Haloalkane dehalogenase LinB) were most abundant after phagocytosis of M. tuberculosis K-strain. This approach provides a method to determine specific proteins that may have critical roles in tuberculosis pathogenesis.

• Korean Journal of Veterinary Research
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• v.36 no.1
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• pp.31-38
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• 1996

1. The sixty raised shepherd and sixty-five inhoused pet dogs in the regions of Daejon and Cheongju were subjected to investigate the TB infection by means of BCG and X-ray diagnosis. The 5 out of 65 inhoused pet and 7 out of 60 shepherd dogs were observed to be infected with TB, respectively. However, none of Mycobacterium species were detected from lung tissues of 4-slaughtered dogs showing BCG positive reaction. 2. The rats were first inoculated with 0.1ml BCG, and then 0.1ml M bovis suspended solution($1{\times}10^5$ organisms/0.2ml) 3weeks later. After 5 months, the animals were killed. The pathohistological results from both groups, TB inoculated and BCG treated groups, were observed on the surface of lung. Furthermore, the severe pathological lesion in the Iung was observed in M bovis inoculated group compared to BCG treated group. 3. The slight macrophage invasion and granuloma formation in the lung from BCG treated group were observe individually. However, it was confirmed that the lung from M bovis treated group was invaded by the macrophages and neutrophils combined with the granuloma formation. 4. When the numbers of the total cells taken from broncho-alvealar fluid in each of mouse from both groups were differentially counted, the number of total cell, neutrophils, and lymphocytes from M bovis treated group were significantly increase compared with those of BCG treated group. 5. Although there were nearly no response of the alveolar macrophages to CSF in serum obtained from control group, those from M boris treated group were significantly proliferated.

• Korean Journal of Veterinary Research
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• v.29 no.3
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• pp.283-289
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• 1989

In order to measure in vitro cell mediated immunity in the guinea pigs sensitized with the killed bacilli of Mycobacterium bovis ($AN_5$), M avium (serotype 2), M tuberculosis and M intracellulare (serotype 8), leukocyte adherence inhibition (LAI) test was established using the antigens of purified protein derivatives (PPD) tuberculin. By using LAI test, specificity of cell-mediated immune responses of the guinea pigs inoculated with various Mycobacterium spp was investigated, and comparison between values of LAI and skin test was also made to evaluate the specificity of the newly designed test. The results obtained throughout the experiments were summarized as follows; 1. The optimal concentration of PPD antigens for LAI test was 1 to 2mg per ml of medium. 2. When the leukocytes of guinea pigs sensitized with both M bovis($AN_5$) and M avium (serotype 2) for 2 to 8 weeks were incubated with homologous or heterologous PPD antigens, mean values of LAI test were $61.2{\pm}11.2$ and $65.6{\pm}5.1%$ in homologous PPD antigens respectively, while $30.0{\pm}3.7$ and $32.8{\pm}5.7%$ in heteNlogous PPD antigens, showing the prominently high value of LAI in the homologous syst,em (p<0.01). 3. When the leukocytes of guinea pigs sensitized with both M tuberculosis and M intracellulare (serotype 8) for 2 to 8 weeks were incubated with homologous and heterologous PPD antigens, mean values of LAI test were $67.9{\pm}2.9$ and $66.9{\pm}5.0%$ in homologous PPD antigens, while $27.4{\pm}7.4$ and $24.4{\pm}7.1%$ in heterologous PPD antigens, showing the prominently high value of LAI in the homologous system (p<0.01). 4. Comparing with the specificity of LAI and skin tests on the basis of the value obtained from the homologous system, deviation of reaction was revealed to be 49.5 to 100.2 in LAI test, and -15.9 to 52.0 in skin test.