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At least 1,531 records · Page 85Linked to original sources
PubMed1999

Presence of eubacteria in biopsies from Crohn's disease inflammatory lesions as determined by 16S rRNA gene-based PCR.

The aim of this study was to search for putative microbial agents in Crohn's disease (CD) tissues by bacterial broad-range 16S rDNA PCR combined with genus- and species-specific DNA hybridisation analysis. Biopsies taken both surgically and endoscopically from the terminal ileum of 11 CD patients and 11 control patients were investigated. Significant amounts of eubacteria were demonstrated in biopsies taken endoscopically from both affected and unaffected individuals; the biopsies taken at surgery from control patients were negative. Three of five biopsies taken surgically from CD patients harboured Helicobacter spp.-, Mycobacterium paratuberculosis-, Listeria monocytogenes- and Escherichia coli-like 16S rDNA sequences. These findings show the importance of the sampling method chosen when combined with molecular typing of eubacteria in intestinal tissues. The mixed bacterial flora found in the surgical biopsies from CD patients supports the idea that the enteric microflora enters primary lesions where secondary bacterial colonisers may elicit a chronic inflammatory syndrome.

Adolescent↗
PubMed1994

Nucleotide sequence and secondary structures of precursor 16S rRNA of slow-growing mycobacteria.

Slow-growing mycobacteria have a single ribosomal RNA (rrn) operon, with the genes for 16S, 23S and 5s rRNA being present in that order. The transcription start site of the rrn operon of Mycobacterium tuberculosis was identified in Escherichia coli. PCR methodology was used to amplify parts of the rrn operon, namely the leader region and the spacer-1 region separating the 16S rRNA and 23S rRNA genes of Mycobacterium avium, Mycobacterium paratuberculosis, Mycobacterium intracellulare, 'Mycobacterium lufu', Mycobacterium simiae and Mycobacterium marinum. The amplified DNA was sequenced. The sequence data, together with those obtained previously for Mycobacterium leprae and M. tuberculosis, were used to identify putative antitermination signals and RNase III processing sites within the leader region. Notable features include a highly conserved Box B element and a sequence of 31 nucleotides which is common to all eight slow-growers which were scrutinized. A secondary structure for mycobacterial precursor-16S rRNA was devised, based on sequence homologies and homologous nucleotide substitutions. The 18 nucleotides at the 5'-end of spacer-1 have the capacity of binding sequences close to the 5'- and 3'-ends of mature 16S rRNA, suggesting that secondary structure is important to the maturation process. All the slow-growers, including M. leprae, conform to the same scheme of secondary structure. The scheme proposed for M. tuberculosis is a variant of the main theme. The leader and spacer sequences may prove a useful supplement to 16S rRNA sequences in establishing phylogenetic relationships between very closely related species. 'M. lufu' appears to be a close relative of M. intracellulare.

Base Sequence↗
PubMed1995

Recombinant BCG expressing the leishmania surface antigen Gp63 induces protective immunity against Leishmania major infection in BALB/c mice.

We have cloned and expressed the gp63 gene of Leishmania major in BCG to develop a recombinant vaccine against zoonotic cutaneous leishmaniasis. Two different expression systems were investigated. The first system consists of pAN, a Mycobacterium paratuberculosis promoter, which drives expression of ORF2, an open reading frame in IS900. This system allows the production of heterologous polypeptides as hybrids with the ORF2 gene product. The second expression system relies on the production of antigenic fragments as fusion proteins with the N-terminal region of Mycobacterium fortuitum beta-lactamase. Both constructs resulted in the production of Gp63 in BCG. The ability of the two recombinant BCG strains to induce protective immunity against a challenge with L. major amastigotes was evaluated after vaccination of susceptible (BALB/c), and resistant (C57BL/6) mice. Recombinant BCG producing Gp63 as a hybrid protein with the N-terminal region of the beta-lactamase elicited significant protection against a challenge with L. major in BALB/c-immunized mice.

Animals↗
PubMed1982

Numerical analysis of the taxonomy of Nocardiae and Rhodococci.

The genera Nocardia and Rhodococcus were clearly differentiated in the present study. Eleven characteristics were shown to be useful for differentiation between these two genera. Nocardia asteroides sunsu stricto previously defined by Tsukamura was divided into two taxa. One contained the type strain and was considered to retain the name Nocardia asteroides in a new sense. Another was named in the present study as Nocardia nova sp. nov. Tsukamura. The type strain of this species is ATCC 33726. The following seven characters were useful for differentiating N. nova from newly defined N. asteroides: 1) arylsulfatase activity after 14 days; 2) catalase activity (semiquantitative); 3) beta-esterase activity; 4) pyrazinamidase activity; 5) utilization of citrate as a sole source of carbon; 6) utilization of 2,3-butylene glycol as a sole carbon source; and 7) resistance to 5-fluorouracil (20 micrograms/ml). The name Nocardia farcinica for Tsukamura's Kyoto-I group should be rejected. This taxon has been named Nocardia paratuberculosis sp. nov. Tsukamura. The type strain is ATCC 23826. Three new species of the genus Rhodococcus were proposed: Rhodococcus aichiensis sp. nov. Tsukamura (type strain, ATCC 33611); Rhodococcus chubuensis sp. nov. Tsukamura (type strain, ATCC 33609); Rhodococcus obuensis sp. nov. Tsukamura (type strain, ATCC 33610).

Actinomycetales↗
PubMed1987

The use of DNA probes identifying restriction-fragment-length polymorphisms to examine the Mycobacterium avium complex.

DNA probes were used to identify restriction-fragment-length polymorphisms (RFLPs) in DNA samples, demonstrating that the Mycobacterium avium complex could be clearly divided into M. avium and Mycobacterium intracellulare strains. Less than 2% DNA base substitution was found between M. avium strains, whereas the M. intracellulare strains had greater than 15% base substitution. The Johne's disease bacillus, Mycobacterium paratuberculosis (American type strain), was found to be distinguishable from the M. avium complex serotypes examined. Strain 18 was found to be identical to M. avium. The rat leprosy bacillus, Mycobacterium lepraemurium, was found to be very closely related, but not identical, to M. avium.

DNA, Bacterial↗
PubMed1991

IS901, a new member of a widespread class of atypical insertion sequences, is associated with pathogenicity in Mycobacterium avium.

An insertion sequence (IS901), found in pathogenic strains of Mycobacterium avium, but absent in M. avium complex isolates from patients with acquired immune deficiency syndrome (AIDS), has been isolated and sequenced. This insertion element has a nucleotide sequence of 1472 bp, with one open reading frame (ORF1), which codes for a protein of 401 amino acids. The amino acid sequence, terminal ends and target site of IS901 are similar to those of IS900, present in Mycobacterium paratuberculosis. However, the DNA sequences of these two IS elements exhibit only 60% homology, compared to a DNA homology of 98% between their respective hosts. IS901, like IS900, appears to belong to a family of related insertion elements present in actinomycetes and other bacteria. M. avium strains containing IS901 were found to be more virulent in mice than closely related strains lacking IS901. IS901 may be a useful tool for the study of the genetics of virulence in the M. avium complex and for obtaining stable integration of foreign genes into mycobacteria.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Characterization of IS1110, a highly mobile genetic element from Mycobacterium avium.

A highly mobile insertion sequence designated IS1110 was detected in Mycobacterium avium strain LR541 following an observed increase in size of the plasmid pLR20. Genomic libraries of M. avium strains carrying either parental pLR20 or the modified plasmid (pLR20') were constructed and the sequence of the relevant clones was determined to characterize the insertion sequence and the target region. IS1110 is a 1457 bp element lacking terminal inverted repeats, and is related to IS900 (from Mycobacterium paratuberculosis), IS901 and IS902 (from M. avium) and to IS116 (from Streptomyces clavuligerus). LR541 carries several copies of IS1110. Individual colonies from the same plate show differences in Southern blot patterns when tested with an IS1110-derived probe; the ability to detect transposition events in random colonies, without any selection pressure, indicates an exceptionally high degree of mobility, which will be invaluable for transposon mutagenesis. Analyses of M. avium isolates from human, veterinary, and environmental sources showed that IS1110-hybridizing sequences are present in some M. avium isolates but they were not detected in strains of other mycobacterial species. The polymorphism exhibited in M. avium isolates suggests that this element may be useful for molecular epidemiological studies of M. avium infections.

Amino Acid Sequence↗
PubMed1988

The killing of tumour cell targets coupled to tuberculin (PPD) by human and murine PPD-reactive T helper clones. I. PPD specificity of killing.

This paper reports on the characteristics of killing by a human and a murine tuberculin (PPD)-specific T helper clone of targets to which PPD was attached via the lectin concanavalin A (Con A). The killing was specific for PPD from M. tuberculosis; and targets coupled to Con A alone or to PPD from M. paratuberculosis were not killed. Target cells carrying Con A-PPD were more effectively lysed than PPD-pulsed cells. This form of lymphocyte killing, though highly significant, was inefficient. Maximum killing of PPD carrying targets was 30-40% at effector to target ratios of 20:1 and at 16 h. Cells carrying 2 x 10(6) molecules of PPD and less than 1.5 x 10(6) molecules Con A per cell were killed most efficiently. A major distinction between this helper T cell killing and that mediated by cytotoxic T cells was that both TH clones displayed bystander lysis and killed PPD uncoupled targets when these were cultured with syngeneic PPD-bound targets. This suggests that the mechanism of cytotoxicity may involve soluble mediators.

Animals↗
PubMed1994

Blood serum transferrin concentration in cattle in various physiological states, in veal calves fed different amounts of iron, and in cattle affected by infectious and non-infectious diseases.

Transferrin (Tf) concentrations were determined in cattle in various physiological states, in energy-deficient (ketotic) cows, in situations of several acute and chronic infections, after endotoxin administration and in animals with bovine leucocyte adhesion deficiency (BLAD). Tf concentrations varied between 1.5 and 8.5 g/l and in healthy animals were in the range of 2.0 and 6.6 g/l. Tf concentrations in adult animals were smaller than in young animals and increased in veal calves with iron deficiency above 8 g/l, resulting in a negative correlation between Hb and Tf. In veal calves total iron binding capacity (TIBC) and Tf concentration were rather closely correlated (r = 0.63). Chronic infectious diseases (such as paratuberculosis) were characterized by relatively low Tf levels (below 2 g/l), while during acute infections, after endotoxin-administration and during ketosis Tf concentrations were not changed.

Animals↗
PubMed1997

Effects of diets with graded levels of naturally deoxynivalenol-contaminated oats on immune response in growing pigs.

A trial was conducted to evaluate the effect of including different levels of deoxynivalenol (DON)-contaminated oats in the complete diets of growing pigs on immune response and performance. The diets contained 0.6, 1.8 and 4.7 mg DON/kg, and both restricted and ad libitum feeding were used. Performance was recorded as weight gain, feed intake, efficiency of feed utilization and carcass quality. Immune response parameters recorded included primary and secondary antibody titres after injections of five different antigens: Human serum albumin (HSA), sheep red blood cells (SRBC), paratuberculosis vaccine (MPT), tetanus toxoid (TT) and diphteria toxoid (DT). A johnin test was also performed. Lymphocyte stimulation response was measured with three different mitogens (PWM, ConA and PHA). A significant, DON dose-dependent reduction in secondary antibody response to tetanus toxoid was observed. A slightly higher mitogen response after PHA stimulation in lymphocytes from the medium and high DON groups compared to the low DON group after 9 weeks was considered inconclusive. No other indication of dose-dependent immune response inhibition or stimulation was found. Significantly reduced feed intake with increased levels of DON was observed in groups fed restricted rations according to weight, but not in animals fed ad libitum.

Animals↗
PubMed1994

Specificity of the immunohistochemical demonstration of mycobacterial antigens.

The specificity of immunohistological reactions of commercial polyclonal antibodies to antigens of various mycobacteria (Mycobacterium bovis, Mycobacterium duvalii and Mycobacterium paratuberculosis) was tested in connection with a number of pathogens and with the help of the indirect immunoperoxidase technique. Positive reactions occurred with various mycobacterial antigens and some Gram-positive bacteria and fungi. These investigations indicate that when the above antibodies are used in histopathological diagnostics, false-positive results can be expected.

Animals↗
PubMed1996

Does Crohn's disease need differentiation from tuberculosis?

Crohn's disease (CD) and tuberculosis (TB) of the gastrointestinal tract pose major diagnostic problems for clinicians where these conditions coexist. Clinically and radiologically, the diseases are similar. In the West, TB is considered in the differential diagnosis of all suspected cases of CD, particularly among Asian migrants. Earlier age of presentation, perianal disease and enteric fistulae favour a diagnosis of CD. Aphthoid ulceration, pseudopolyps and filiform mucosa at endoscopy are suggestive of CD and a negative tuberculin test us useful. The final diagnosis depends largely on histopathology and the presence or absence of acid fast bacilli. Tuberculosis is more common in developing countries and intestinal TB frequently coexists with pulmonary tuberculosis. TB is known to affect all age groups and fistulous communication, although uncommon, does occur. In addition to radiology and endoscopy, laparotomy may be required to establish the diagnosis. In developing countries, CD is uncommon and remains largely a diagnosis of exclusion. A trial of anti-tuberculosis therapy may often be prescribed before definitely diagnosing CD. The development of molecular biology techniques had led to a revival of interest in mycobacteria as a possible aetiological agent in CD. DNA from Mycobacterium paratuberculosis and Mycobacterium kansaii have both been identified in CD cases but the significance of this finding has not been established. However, in the near future polymerase chain reaction will become increasingly useful in differentiating CD from intestinal TB because it allows the amplification and identification of very small quantities of mycobacterium DNA.

Crohn Disease↗
PubMed1990

Phylogenetic analysis and identification of different serovars of Mycobacterium intracellulare at the molecular level.

Comparative 16S rRNA sequencing was used to infer the phylogenetic relationship among different serovars of the Mycobacterium avium-M. intracellulare complex as well as to define signature nucleotides characteristic for different serovars. In general, the groups defined by rRNA sequencing reflect the classification obtained with sensitin tests and pathogenicity examinations in chickens. Unique 16S rRNA sequence patterns could be defined for (1) M. avium, (2) M. intracellulare serovars 4, 5, 6, 8, 9, 10 and 11, (3) M. intracellulare serovars 12, 13, 14, 15, 17, 19 and 20, (4) M. intracellulare serovar 7 and (5) M. intracellulare serovar 18. Phylogenetically, groups 1 and 2 on one hand and groups 3, 4 and 5 on the other hand each share a common ancestor. M. paratuberculosis was indistinguishable from M. intracellulare serovars 4, 5, 6, 8, 9, 10 and 11 by this kind of analysis.

Base Sequence↗
PubMed2000

In vitro activities of the ketolides telithromycin (HMR 3647) and HMR 3004 compared to those of clarithromycin against slowly growing mycobacteria at pHs 6.8 and 7.4.

The in vitro activities of HMR 3647 (telithromycin) and HMR 3004, two novel semisynthetic ketolides, were investigated and compared with that of the reference macrolide drug, clarithromycin, against 34 strains of slowly growing mycobacteria at pHs 6.8 and 7.4, as determined radiometrically. The MICs at pH 7.4 were about 1 to 2 dilutions lower than those observed at pH 6.8. In terms of the highest to the lowest activity, the three antibiotics could be classified as follows: clarithromycin > HMR 3004 > HMR 3647. Among the species tested, Mycobacterium bovis BCG, M. ulcerans, M. avium, and M. paratuberculosis were moderately susceptible to HMR 3004 and HMR 3647 (MICs at pH 7.4, < or =5.0 and < or =20.0 microg/ml, respectively, versus < or =1.25 microg/ml for clarithromycin), whereas M. tuberculosis, M. africanum, M. bovis, and M. simiae were resistant (MICs, > or =10.0 and > or =40.0 microg/ml, respectively, at pH 7.4). Although not more active than clarithromycin in vitro, the high level of intracellular accumulation of the two ketolides inside phagocytes warrants further screening in experimental animal models.

Anti-Bacterial Agents↗
PubMed2001

In vitro activity of a novel antimycobacterial compound, N-octanesulfonylacetamide, and its effects on lipid and mycolic acid synthesis.

beta-Sulfonyl carboxamides have been proposed to serve as transition-state analogues of the beta-ketoacyl synthase reaction involved in fatty acid elongation. We tested the efficacy of N-octanesulfonylacetamide (OSA) as an inhibitor of fatty acid and mycolic acid biosynthesis in mycobacteria. Using the BACTEC radiometric growth system, we observed that OSA inhibits the growth of several species of slow-growing mycobacteria, including Mycobacterium tuberculosis (H37Rv and clinical isolates), the Mycobacterium avium complex (MAC), Mycobacterium bovis BCG, Mycobacterium kansasii, and others. Nearly all species and strains tested, including isoniazid and multidrug resistant isolates of M. tuberculosis, were susceptible to OSA, with MICs ranging from 6.25 to 12.5 microg/ml. Only three clinical isolates of M. tuberculosis (CSU93, OT2724, and 401296), MAC, and Mycobacterium paratuberculosis required an OSA MIC higher than 25.0 microg/ml. Rapid-growing mycobacterial species, such as Mycobacterium smegmatis, Mycobacterium fortuitum, and others, were not susceptible at concentrations of up to 100 microg/ml. A 2-dimensional thin-layer chromatography system showed that OSA treatment resulted in a significant decrease in all species of mycolic acids present in BCG. In contrast, mycolic acids in M. smegmatis were relatively unaffected following exposure to OSA. Other lipids, including polar and nonpolar extractable classes, were unchanged following exposure to OSA in both BCG and M. smegmatis. Transmission electron microscopy of OSA-treated BCG cells revealed a disruption in cell wall synthesis and incomplete septum formation. Our results indicate that OSA inhibits the growth of several species of mycobacteria, including both isoniazid-resistant and multidrug resistant strains of M. tuberculosis. This inhibition may be the result of OSA-mediated effects on mycolic acid synthesis in slow-growing mycobacteria or inhibition via an undescribed mechanism. Our results indicate that OSA may serve as a promising lead compound for future antituberculous drug development.

Acetamides↗
PubMed2004

Analysis of possible factors affecting the specificity of the gamma interferon test in tuberculosis-free cattle herds.

Bovine tuberculosis (TB) is still a zoonotic problem in the world. Despite the fact that eradication programs for bovine TB are being implemented in many countries, it remains a public health problem. These programs are mainly based on a single intradermal tuberculin test using bovine tuberculin purified protein derivative (PPD), isolation, and slaughtering of infected animals. The aim of this study was to assess the specificity of the gamma interferon (IFN-gamma) test in TB-free cattle herds, by using not only Australian tuberculins but also tuberculins produced at our institute, and to correlate the response with the type of production (beef cattle, dairy cattle, and a dual-purpose breed), the housing system, and the age of the animals. We studied 800 animals selected from 20 TB- and paratuberculosis-free herds. The animals were tested in parallel, after stimulation with Australian tuberculins and tuberculins produced at our institute, by using the skin test and two IFN-gamma assays. The results of this trial showed that the specificity of the IFN-gamma test is higher than that of the skin test (96.8%) and ranges from 97.3% (using only Australian tuberculins) to 98.6% (using tuberculins produced at our institute). We found that different categories of cattle could influence the specificity of the skin test but that these differences tended to be reduced in the IFN-gamma assay, especially when Italian PPDs were used.

Age Factors↗
PubMed1989

Production of a monoclonal antibody specific for Mycobacterium avium and immunological activity of the affinity-purified antigen.

Three hybridomas which secrete antibodies to Mycobacterium avium were obtained by the fusion of p3u1 myeloma cells with spleen cells of mice immunized with M. avium culture sonicate. The reactivity of these monoclonal antibodies was determined in 16 species of mycobacteria by an enzyme-linked immunosorbent assay. An antibody, designated Avi-3, reacted only with M. avium and not with the reference strains of the other 15 species of mycobacteria tested, including M. intracellulare, M. paratuberculosis, and M. lepraemurium. Specificity of the antibody was confirmed by assay, using a specific DNA probe of M. avium complex in 29 M. avium complex isolates. An antigen was purified from M. avium culture sonicate on a monoclonal antibody Avi-3-coupled affinity column. The purified antigen gave a single band (molecular size, about 27 kilodaltons) upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antigen Avi-3 showed strong skin test activity in guinea pig preimmunized with heat-killed M. avium but not in those sensitized with heat-killed M. intracellulare or M. bovis BCG. Purified protein derivative elicited positive skin reactions in all the immunized guinea pigs. When heat-killed M. avium was used as the immunogen, strong lymphoproliferative responses were observed in cultures stimulated with the antigen Avi-3. These results suggest that M. avium-specific antigen Avi-3 may facilitate the diagnosis of mycobacterial infections.

Animals↗
PubMed1999

Protection against Mycobacterium avium by DNA vaccines expressing mycobacterial antigens as fusion proteins with green fluorescent protein.

Mycobacterium avium causes disseminated disease in humans with AIDS, paratuberculosis in ruminants, lymphadenopathy in swine, and tuberculosis in birds. We constructed DNA vaccines expressing mycobacterial antigens as fusion proteins with enhanced green fluorescent protein (EGFP). Plasmids p65K-EGFP, p85A-EGFP, and p85B-EGFP expressed the M. avium 65-kDa antigen, the Mycobacterium bovis BCG 85A antigen, and the M. avium 85B antigen, respectively, as EGFP fusion proteins. We visualized protein expression directly in cultured murine fibroblasts and intact muscle. p65K-EGFP expressed fusion protein in a diffuse cytoplasmic pattern, and p85A-EGFP and p85B-EGFP produced a speckled pattern. We vaccinated C57BL/6 mice with three doses of plasmid DNA and then challenged them intraperitoneally with M. avium. Negative controls received saline, and positive controls received one dose of BCG vaccine. Mice in all groups developed disseminated infection with a high burden of organisms. Compared to negative controls, mice vaccinated with p85A-EGFP had an eightfold reduction in spleen M. avium CFU at 4 weeks after infection and a fourfold reduction at 8 weeks, reductions similar to those generated by BCG vaccine. Mice vaccinated with p65K-EGFP had a fourfold CFU reduction at 4 weeks and no effect at 8 weeks. This is the first report of DNA vaccines expressing foreign antigens as fusion proteins with EGFP and the first report of successful DNA vaccination against M. avium.

3T3 Cells↗