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PubMed2005

Identification of Mycobacterium avium genes that affect invasion of the intestinal epithelium.

Invasion of intestinal mucosa of the host by Mycobacterium avium is a critical step in pathogenesis and likely involves several different bacterial proteins, lipids, glycoproteins, and/or glycolipids. Through the screening of an M. avium genomic library in Mycobacterium smegmatis, we have identified a number of M. avium genes that are associated with increased invasion of mucosal epithelial cells. In order to further investigate these genes, we cloned six of them into a plasmid downstream of a strong mycobacterial promoter (L5 mycobacterial phage promoter), resulting in constitutive expression. Bacteria were then evaluated for increased expression and examined for invasion of HT-29 intestinal epithelial cells. The genes identified encode proteins that are similar to (i) M. tuberculosis coenzyme A carboxylase, (ii) M. tuberculosis membrane proteins of unknown function, (iii) M. tuberculosis FadE20, (iv) a Mycobacterium paratuberculosis surface protein, and (v) M. tuberculosis cyclopropane fatty acyl-phopholipid synthase. The constitutive expression of these genes confers to M. avium the ability to invade HT-29 intestinal epithelial cells with a severalfold increase in efficiency compared to both the wild-type M. avium and M. avium containing the vector alone. Using the murine intestinal ligated loop model, it was observed that the constitutive expression of M. avium proteins has a modest impact on the ability to enter the intestinal mucosa when compared with the wild-type control, suggesting that under in vivo conditions these genes are expressed at higher levels. Evaluation of the expression of these invasion-related genes indicated that under conditions similar to the intestinal lumen environment, the genes identified are upregulated. These data suggest that invasion of the intestinal mucosa is an event that requires the participation of several bacterial factors and the expression of the genes that encode them is less observed under standard laboratory growth conditions.

Cell Line↗
PubMed2005

Bovine NK cells can produce gamma interferon in response to the secreted mycobacterial proteins ESAT-6 and MPP14 but not in response to MPB70.

Bovine NK cells have recently been characterized and the present study describes the interaction between NK cells, antigen-presenting cells, and secreted mycobacterial proteins. Gamma interferon (IFN-gamma) production by NK cells was seen in approximately 30% of noninfected calves in response to the Mycobacterium tuberculosis complex-specific protein ESAT-6, MPP14 from Mycobacterium avium subsp. paratuberculosis, and purified protein derivative (PPD) from M. tuberculosis. In contrast, no response was induced by MPB70, which is another M. tuberculosis complex-specific secreted antigen. The production of IFN-gamma by NK cells in whole blood in response to ESAT-6 and MPP14 was demonstrated using intracellular staining together with surface labeling for the NK cell-specific receptor, NKp46, or CD3. Furthermore, the depletion of NK cells from peripheral blood mononuclear cells completely abolished the IFN-gamma production. The response was mediated through stimulation of adherent cells and was largely independent of contact between adherent cells and the NK cells. Neutralization of interleukin-12 only partly inhibited IFN-gamma production, showing that other cytokines were also involved. The demonstration of NK cell-mediated IFN-gamma production in young cattle provides an explanation for the nonspecific IFN-gamma response frequently encountered in young cattle when using the IFN-gamma test in diagnosis of mycobacterial infections.

Animals↗
PubMed1990

The division between fast- and slow-growing species corresponds to natural relationships among the mycobacteria.

Comparative 16S rRNA sequencing was used to infer the phylogenetic relationships among selected species of mycobacteria and related organisms. The phylogeny inferred reflects the traditional classification, with major branches of the phylogenetic tree in general correspondence to the four Runyon groups and with numerical classification analyses. All the mycobacterial species compared, with the exception of M. chitae, are closely related (average similarity values greater than 95%). The slow growers form a coherent line of descent, distinct from the rapid growers, within which the overt pathogens are clustered. The distant relationship between M. chitae and the remaining mycobacteria suggests that this organism is incorrectly classified with the mycobacteria. M. paratuberculosis 18 was indistinguishable from M. avium-M. intracellulare-M. scrofulaceum serovar 1 by this analysis.

Base Sequence↗
PubMed1984

Characteristics of an unclassified Mycobacterium species isolated from patients with Crohn's disease.

The characteristics of an unclassified Mycobacterium sp. isolated from three patients with Crohn's disease are presented. The organism is extremely fastidious and mycobactin dependent and may require up to 18 months of incubation for primary isolation. Colony morphology is rough. Characteristics are unlike those of any presently defined species. The isolates produced postive niacin, catalase, and 2-week arylsulfatase reactions and were susceptible to neotetrazolium chloride (1:40,000), streptomycin (2 micrograms/ml), and rifampin (0.25 micrograms/ml). Chromogenicity, nitrate reduction, quantitative catalase, Tween hydrolysis, urease, tellurite reduction, pyrazinamidase, and 3-day arylsulfatase tests were negative, and the isolates were resistant to thiophene-2-carboxylic acid hydrazide (10 micrograms/ml) and isoniazid (10 micrograms/ml). Optimum growth in broth was determined to be in 7H9 medium with Dubos oleic albumin complex, Tween 80, and mycobactin J at 37 degrees C without CO2 or agitation and in low medium depth. This Mycobacterium sp. may be a subspecies or biovariant of Mycobacterium paratuberculosis, or it may represent a new species of Mycobacterium. It is suggested that this Mycobacterium sp. may play an etiological role in some cases of Crohn's disease.

Adolescent↗
PubMed1990

Detection and identification of mycobacteria by amplification of rRNA.

Oligonucleotides specific at a genus, group, or species level were defined by a systematic comparison of small-subunit rRNA sequences from Mycobacterium tuberculosis, M. bovis, M. africanum, M. bovis BCG, M. avium, M. kansasii, M. marinum, M. gastri, M. chelonae, M. smegmatis, M. terrae, M. nonchromogenicum, M. xenopi, M. malmoense, M. szulgai, M. scrofulaceum, M. fortuitum, M. gordonae, M. intracellulare, M. simiae, M. flavescens, M. paratuberculosis, M. sphagni, M. cookii, M. komossense, M. phlei, and M. farcinica. On the basis of the defined oligonucleotides, the polymerase chain reaction technique was explored to develop a sensitive taxon-specific detection system for mycobacteria. By using M. tuberculosis as a model system, fewer than 10 bacteria could be reliably detected by this kind of assay. These results suggest that amplification of rRNA sequences by the polymerase chain reaction may provide a highly sensitive and specific tool for the direct detection of microorganisms without the need for prior cultivation.

Base Sequence↗
PubMed1993

Identification of mycobacteria from animals by restriction enzyme analysis and direct DNA cycle sequencing of polymerase chain reaction-amplified 16S rRNA gene sequences.

Two methods, based on analysis of the polymerase chain reaction-amplified 16S rRNA gene by restriction enzyme analysis (REA) or direct cycle sequencing, were developed for rapid identification of mycobacteria isolated from animals and were compared to traditional phenotypic typing. BACTEC 7H12 cultures of the specimens were examined for "cording," and specific polymerase chain reaction amplification was performed to identify the presence of tubercle complex mycobacteria. Combined results of separate REAs with HhaI, MspI, MboI, and ThaI differentiated 12 of 15 mycobacterial species tested. HhaI, MspI, and ThaI restriction enzyme profiles differentiated Actinobacillus species from mycobacterial species. Mycobacterium bovis could not be differentiated from M. bovis BCG or Mycobacterium tuberculosis. Similarly, Mycobacterium avium and Mycobacterium paratuberculosis could not be distinguished from each other by REA but were differentiated by cycle sequencing. Compared with traditional typing, both methods allowed rapid and more accurate identification of acid-fast organisms recovered from 21 specimens of bovine and badger origin. Two groups of isolates were not typed definitively by either molecular method. One group of four isolates may constitute a new species phylogenetically very closely related to Mycobacterium simiae. The remaining unidentified isolates (three badger and one bovine) had identical restriction enzyme profiles and shared 100% nucleotide identify over the sequenced signature region. This nucleotide sequence most closely resembled the data base sequence of Mycobacterium senegalense.

Animals↗
PubMed1993

Genus-specific polymerase chain reaction for the mycobacterial dnaJ gene and species-specific oligonucleotide probes.

Identification of tuberculous and nontuberculous mycobacteria by biochemical methods is a long-term process that takes up to 8 weeks for completion and requires expertise to interpret the results. In order to detect and differentiate the major pathogenic mycobacterial species, we developed genus-specific primers that amplify the dnaJ gene from the broad spectrum of mycobacterial species and determined the nucleotide sequences within the dnaJ genes from 19 mycobacterial species (Mycobacterium tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. microti, M. kansasii, M. marinum, M. gastri, M. simiae, M. scrofulaceum, M. szulgai, M. gordonae, M. avium, M. intracellulare, M. xenopi, M. fortuitum, M. chelonei, M. haemophilum, and M. paratuberculosis). On the basis of the dnaJ gene sequences, we developed dot blot hybridization analysis with species-specific oligonucleotide probes for the M. tuberculosis complex. M. avium, M. intracellulare, and M. kansaii, allowing a rapid identification of these species following polymerase chain reaction for the dnaJ gene. We conclude that polymerase chain reaction with the genus-specific primer that amplifies the dnaJ genes and subsequent dot blot analysis with species-specific oligonucleotide probes are most useful for differential diagnosis of tuberculosis and nontuberculous mycobacterial infections.

Base Sequence↗
PubMed1993

Differentiation of mycobacteria on the basis of chemotype profiles by using matrix solid-phase dispersion and thin-layer chromatography.

Because of the rising incidence of clinical mycobacterial infections and the difficulty in identification and characterization of mycobacteria at the subspecies and serovar levels, a technique for differentiation that could be performed quickly and with relatively little equipment and expense was developed. Lysis and fractionation of mycobacteria by matrix solid-phase dispersion followed by thin-layer chromatography were used to produce chemotype profiles of the lipid and glycolipid components of each isolate. Organisms tested included Mycobacterium scrofulaceum, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium flavescens, Mycobacterium kansasii, Mycobacterium bovis, 11 isolates of Mycobacterium gordonae, 10 serovars of Mycobacterium avium, and four strains of Mycobacterium paratuberculosis. A relative retention (Rx) value was established for each visible band on the chromatographs by comparison with a band common to all organisms tested. The chemotype profiles produced were different for every separate species and serovar sampled. These findings suggest that matrix solid-phase dispersion and subsequent thin-layer chromatography may have the sensitivity and flexibility to characterize and identify mycobacteria at the species and subspecies levels and potentially to identify clinical isolates of mycobacteria that have been difficult to identify by standard means. The technique has certain advantages over current biochemical, immunologic, and genetic methods because it is relatively simple to perform, is inexpensive, and requires a small amount of bacterial sample.

Bacterial Typing Techniques↗
PubMed1993

Rapid detection and identification of Mycobacterium avium by amplification of 16S rRNA sequences.

An assay that is based on the amplification of 16S rRNA sequences and that was initially developed to detect Mycobacterium paratuberculosis in cattle was used to test 20 serotypes of the Mycobacterium avium complex (MAC) and atypical mycobacterial species not belonging to MAC. Only serotypes 1 to 6 and 8 to 11, designated M. avium, were detected by the assay, indicating that it can be used for the rapid detection and identification of M. avium. The results of the assay for clinical samples from animals suspected of having mycobacterial infections indicated that it can also be used directly on clinical samples.

Animals↗
PubMed2000

Distinct differences in repertoires of low-molecular-mass secreted antigens of Mycobacterium avium complex and Mycobacterium tuberculosis.

Antigens in a 4-week-old culture filtrate (CF) of Mycobacterium avium subsp. avium were separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by Western blotting. The culture had minimal lysis of bacilli, giving a CF preparation consisting mainly of secreted proteins. Comparison with a similar CF of Mycobacterium tuberculosis with almost no contamination with intracellular proteins showed the presence of cross-reactive antigens homologous to the four components of the antigen 85 complex, as well as MPT32. These were major constituents of the M. avium subsp. avium CF. In addition, there were several low-molecular-mass bands (<15 kDa) in both species that did not cross-react with polyclonal and polyvalent rabbit antibodies in Western blotting. Furthermore, these bands were not detected in corresponding sonicate preparations, indicating high localization indexes, which is typical of soluble secreted proteins. A 14-kDa protein was selected for purification and more detailed characterization. The N-terminal amino acid sequence was determined, and a matching gene was found within the genomic sequence of M. avium subsp. avium which was highly homologous to Rv0455c of M. tuberculosis. The gene encoded a signal peptide typical of secreted mycobacterial proteins. A rabbit antiserum was raised against the purified protein, and the antigen was demonstrated by Western blotting in CFs of M. avium subsp. avium, Mycobacterium avium subsp. paratuberculosis, Mycobacterium intracellulare, and Mycobacterium scrofulaceum but was not detected in M. tuberculosis. This is a new example of a highly homologous gene being differentially expressed by different mycobacterial species.

Amino Acid Sequence↗
PubMed2006

Isolation of the genome sequence strain Mycobacterium avium 104 from multiple patients over a 17-year period.

The genome sequence strain 104 of the opportunistic pathogen Mycobacterium avium was isolated from an adult AIDS patient in Southern California in 1983. Isolates of non-paratuberculosis M. avium from 207 other patients in Southern California and elsewhere were examined for genotypic identity to strain 104. This process was facilitated by the use of a novel two-step approach. In the first step, all 208 strains in the sample were subjected to a high-throughput, large sequence polymorphism (LSP)-based genotyping test, in which DNA from each strain was tested by PCR for the presence or absence of 4 hypervariable genomic regions. Nineteen isolates exhibited an LSP type that resembled that of strain 104. This subset of 19 isolates was then subjected to high-resolution repetitive sequence-based PCR typing, which identified 10 isolates within the subset that were genotypically identical to strain 104. These isolates came from 10 different patients at 5 clinical sites in the western United States, and they were isolated over a 17-year time span. Therefore, the sequenced genome of M. avium strain 104 has been associated with disease in multiple patients in the western United States. Although M. avium is known for its genetic plasticity, these observations also show that strains of the pathogen can be genotypically stable over extended time periods.

Bacterial Typing Techniques↗
PubMed2006

Differentiating host-associated variants of Mycobacterium avium by PCR for detection of large sequence polymorphisms.

The Mycobacterium avium species consists of a group of organisms that are genetically related but phenotypically diverse, with certain variants presenting clear differences in terms of their host association and disease manifestations. The ability to distinguish between these subtypes is of relevance for accurate diagnosis and for control programs. Using a comparative genomics approach, we have uncovered large sequence polymorphisms that are, respectively, absent from bird-type M. avium isolates and from cattle types and sheep types of M. avium subsp. paratuberculosis. By evaluating the distribution of these genomic polymorphisms across a panel of strains, we were able to assign unique genomic signatures to these host-associated variants. We propose a simple PCR-based strategy based on these polymorphisms that can rapidly type M. avium isolates into these subgroups.

Animals↗
PubMed1988

Investigation of mycobacteria in Crohn's disease tissue by Southern blotting and DNA hybridisation with cloned mycobacterial genomic DNA probes from a Crohn's disease isolated mycobacteria.

A mycobacterial aetiology for Crohn's disease (CD) has been suggested. Slow growing mycobacteria indistinguishable from M paratuberculosis, the causative agent of enteritis in ruminants (Johne's disease) have been isolated from CD tissues. We have used cloned genomic DNA probes derived from a CD isolated mycobacteria strain Ben, to investigate the presence of mycobacterial DNA sequences in CD tissues. DNA was extracted from total tissue from 17 CD and four control gut specimens. DNA was digested with restriction endonucleases, electrophoresed and transferred to nylon membranes by Southern blotting and hybridised to radiolabelled DNA probes. No mycobacterial DNA was detected in any tissue sample studied. Reconstitution experiments with known numbers of in vitro cultured mycobacteria showed sensitive detection of mycobacterial DNA. DNA extracted from mouse liver, infected with M lepraemurium revealed a strong hybridisation signal and showed the applicability of the experimental approach to the detection of mycobacterial DNA in naturally infected tissues. The results do not provide evidence for the involvement of mycobacteria in the pathogenesis of CD but do not exclude the possibility of low levels of infection in subsets of intestinal cells with spheroplast or cell wall deficient forms of mycobacteria.

Blotting, Southern↗
PubMed1992

Antigen induced suppression in peripheral blood and lamina propria mononuclear cells in inflammatory bowel disease.

Using an autologous system, suppressor cell function to a range of mycobacterial antigens and Kunin antigen of peripheral blood mononuclear cells and lamina propria lymphocytes has been investigated in normal subjects and patients with inflammatory bowel disease. In the peripheral blood there was reduced antigen induced suppression in patients with Crohn's disease in remission to Mycobacterium paratuberculosis, purified protein derivative (PPD), M fortuitum, and Kunin antigens (p less than 0.05). In patients with ulcerative colitis in remission there was reduced antigen induced suppression in the peripheral blood to Kunin antigen (p less than 0.001), M avium (p less than 0.01), M nonchromogenecin, and M fortuitum (p less than 0.05). The phenomenon of antigen induced suppression was largely CD8 dependent, as depleting CD8+ cells reduced the effect and the concentration of soluble CD8 in the culture supernatant was directly related to the suppressor index (r = 0.25, p less than 0.05). These results are likely to be a true reflection of the cell mediated response to antigen as patients with a positive Mantoux skin test have a significantly higher suppressor index to PPD than Mantoux negative subjects (p less than 0.05). These findings may have significance in the aetiopathogenesis of inflammatory bowel disease. However, a similar effect could not be shown in the lamina propria lymphocytes of patients having colectomy for active disease.

Adult↗
PubMed1983

Bacteria in enteric lesions of cattle.

Thirty-nine species of bacteria were isolated from or demonstrated in the abomasal and small and large intestinal mucosa of 23 adult cattle and 41 calves and identified. The bacteria isolated were related to the gross and microscopical lesions. Campylobacters, Clostridium perfringens type A, C sordellii, Actinobacillus lignieresii, Fusobacterium necrophorum, Mycobacterium paratuberculosis, Aeromonas hydrophila and Escherichia coli were all associated with specific lesions. The relationship of other bacteria such as Bacillus licheniformis, Branhamella catarrhalis and Bacteroides vulgatus to lesions in which they were found was discussed. It was concluded that some of the bacteria could be responsible for the lesions in which they were found. However, proof of this supposition could only be obtained by experimental infections of non-immune cattle with pure cultures.

Animals↗
PubMed2005

Survey of zoonoses recorded in Scotland between 1993 and 2002.

All the human and animal laboratory reports of zoonoses sent to Health Protection Scotland between 1993 and 2002 were identified. There were 24,946 reports from veterinary laboratories, and 94,718 (20 per cent) of the 468,214 reports from medical laboratories were considered to be zoonotic. The most common reports of zoonoses from people were Campylobacter, Salmonella, Cryptosporidium and Giardia species and Escherichia coli o157. The most common reports of zoonoses from animals were Salmonella, Cryptosporidium, Chlamydia and Campylobacter species and Mycobacterium avium paratuberculosis. For all the zoonoses in people, the National Health Service Board areas Borders, Dumfries and Galloway, Forth Valley, Grampian, Lanarkshire and Lothian had a higher than expected standardised incidence rate of infection, whereas Ayrshire and Arran, Fife, Greater Glasgow, Shetland, Tayside and Western Isles had a lower than expected rate. The organisms and diseases considered to be new and emerging were Rhodococcus species, Cyclospora cayetanensis, Leishmania species, Pneumocystis carinii (jiroveci) and bovine spongiform encephalopathy/variant Creutzfeldt-Jakob disease.

Animals↗
PubMed2006

Serine threonine protein kinases of mycobacterial genus: phylogeny to function.

Serine/threonine protein kinases (STPKs) are known to act as sensors of environmental signals that thereby regulate developmental changes and host pathogen interactions. In this study, we carried out comparative genome analysis of six completely sequenced pathogenic and nonpathogenic mycobacterial species to systematically characterize the STPK complement of mycobacterium. Our analysis revealed that while Mycobacterium tuberculosis strains have 11 conserved kinases, this number varies from 4 to 24 in other mycobacterial species. pknA, an essential STPK encoding gene, was found to be truncated in the initial analysis of M. avium subsp. paratuberculosis (Map) and M. tuberculosis C genomes. However, resequencing of pknA gene in Map confirmed that the truncation was due to a sequencing error. The conservation of division and cell wall gene cluster involved in cell envelope biosynthesis and cell division, in the vicinity of pknL locus, implicates a possible role of PknL in cell division and envelop biosynthesis. We identified a cyclophilin domain as part of a mycobacterial kinase in Map that suggests a plausible regulation of cyclophilins by phosphorylation. The co-inheritance of pknA, pknB, pknG, and pknL loci across genomes and some unique repertoire of pathogen-specific kinases such as pknI and pknJ of Mtb complex suggest similitude and divergence between pathogenic and nonpathogenic signaling. This study would add another dimension toward identification of physiological substrates and thereby function, while resolving the existing complexities in signaling network between the two domains of life, pathogen and nonpathogen.

Bacterial Proteins↗
PubMed1994

Distribution of anti-CD68 (EBM11) immunoreactivity in formalin-fixed, paraffin-embedded bovine tissues.

A commercially acquired anti-human macrophage antibody (anti-CD68; EBM11) was used in an immunocytochemical technique to detect macrophages in formalin-fixed, paraffin-embedded tissues from cattle, pigs, humans, rats, turkeys, dogs, and cats. In healthy cattle, the antibody labeled alveolar macrophages, pulmonary intravascular cells (presumably intravascular macrophages), and macrophage-like cells in other tissues. In bovine lungs infected with Pasteurella haemolytica, EBM11 antibody labeled 95% of alveolar macrophages and macrophages within alveolar septa but only 0-2% of streaming or "oat" leukocytes. Alveolar macrophages were also stained by EBM11 in pigs but not in rats, turkeys, dogs, and cats. The antibody also stained macrophage aggregates in the mesenteric lymph nodes and intestinal lamina propria of Mycobacterium paratuberculosis-infected cattle. This study shows that the anti-CD68 (EBM11) antibody is a useful marker of macrophages in normal bovine tissues or tissues from areas of acute or chronic inflammation that have been routinely processed. The study also adds strength to the growing evidence suggesting that streaming leukocytes seen in pneumonic pasteurellosis are neutrophils.

Adult↗