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PubMed2003

Infection and the gut.

Gastrointestinal symptoms, including vomiting, are caused by a variety of infective organisms in children, many of which are self-limiting and resolve within a week, but others are potentially much more serious in their consequences. Diarrhea, vomiting and abdominal pain are common but nonspecific symptoms. Investigation is dictated by the likely causative organism, given the age and presentation of the child. The role of bacteria in the pathogenesis of necrotizing enterocolitis, recognition that Yersinia, Campylobacter and Salmonella may produce symptoms difficult to distinguish clinically from appendicitis, the viral causes of idiopathic intussusception, the occurrence of intussusception after administration of rotavirus vaccine, and the evidence incriminating mycobacterium avium subspecies paratuberculosis in the aetiology of Crohn disease are discussed.

Anti-Infective Agents↗
PubMed2000

Evaluation of the specificity of the gamma-interferon test in Italian bovine tuberculosis-free herds.

We investigated the specificity of the gamma-interferon test for bovine tuberculosis (TB) in 1,557 cattle in 30 paratuberculosis-free and officially certified TB-free dairy herds, located in three provinces of the Lombardy Region in Northern Italy. The TB-free status of the herds under examination was further confirmed by the tuberculin skin test, by an antibody assay and by post mortem examination of animals culled from the herds during the study period. The specificity of the gamma-interferon tests after a single test and a double sampling scheme were 88.8% and 95.4%, respectively. After a single test, 11.7% of dubious reactors were also detected, while most cattle (47.4%) were shown to be avian reactors, probably due to contamination from infected birds and/or forage. There was strong evidence that the specificity of the test could be related to the animals' interaction with environmental mycobacteria and/or ageing. To reduce the percentage of nonspecific bovine reactors under alleged TB-free conditions, test procedures might involve the use of more specific antigens and/or different reaction thresholds.

Animals↗
PubMed2001

Dietary effects on the microbiological safety of food.

The high mortality rate associated with human infections caused by Escherichia coli strains of the serotype O157:H7 has brought to public attention the importance of ruminants as reservoirs of food-borne pathogens. In addition to established examples such as salmonella, campylobacter and listeria, recent evidence is emerging of the role of food in the transmission of Helicobacter pylori and Mycobacterium paratuberculosis. Food-borne pathogens harboured by ruminants are spread through shedding in the faeces and subsequent faecal contamination of raw food. Ruminant shedding appears to be affected by diet and, of particular concern, may be increased during fasting regimens imposed during transport to the slaughterhouse. The survival of food-borne pathogens in the ruminant gut is affected by many factors including microbe-microbe interactions, interactions involving plant metabolites and the presence of inhibitory end-product metabolites such as short-chain fatty acids. The potential importance of digesta flow and bacterial detachment in shedding of food-borne pathogens is discussed. Experimental procedures with dangerous pathogens have constraints, particularly in animal experimentation. This situation may be overcome by the use of rumen-simulating fermentors. One such system which, like the natural rumen, has a different turnover rate for solid and liquid digesta, was found to maintain rumen-like variables over an 11 d period. This system may prove useful for the study of dietary effects on food-borne pathogens.

Animals↗
PubMed1996

Attempted definition by immunoblotting of the causes of reactivity in suspected false-positive sera in the Brucella ovis complement fixation test.

Seventy-nine suspected false-positive sera, obtained over 1 year from routine submissions for Brucella ovis serological testing, were used in this study. These sera, which exhibited titres in the complement fixation test, but which because of their epidemiological history and their reactions in the enzyme-linked immunosorbent assay and gel diffusion test were suspected to be false positives, were further analysed by immunoblotting. In blots, using B. ovis antigens, rough lipopolysaccharide was identified as the major, immuno-reactive bacterial component. Antibodies against this macromolecule were present in 46.8% of the suspected false-positive sera. In order to find out if rough lipopolysaccharides from other bacterial species could be the possible cause for the suspected false positivity, 23 sera with highest complement fixation titres were reacted in blots with cell extracts from Escherichia coli, Yersinia enterocolitica, Yersinia pseudotuberculosis, Bortedella bronchiseptica, Actinobacillus seminis, Campylobacter fetus fetus, Campylobacter jejuni, Mycobacterium paratuberculosis, Mycobacterium phlei, Corynebacterium pseudotuberculosis and pure lipopolysaccharides from Escherichia coli and Salmonella typhimurium. Despite high frequencies of antibody reaction with proteins in most of these bacterial cell extracts, which reflect the presence of infections with these bacteria, immuno-staining in the rough lipopolysaccharide region was not observed.

Journal Article↗
PubMed1989

Pulmonary mycosis in farmed deer: allergic zygomycosis and invasive aspergillosis.

During 1988, pulmonary mycosis was diagnosed in four of 116 farmed deer examined on suspicion of tuberculosis. The histopathology showed allergic bronchopulmonary mycosis in a red deer (Cervus elaphus) and the agent was identified as a zygomycete, probably Absidia corymbifera, by immunofluorescence staining. Three fallow deer (Dama dama) had invasive necrotizing mycotic pneumonia and progressive exudative mycotic alveolitis caused by Aspergillus fumigatus. In the red deer, weakness due to paratuberculosis had probably promoted the mycotic infection. The three fallow deer were bred on another farm, where predisposing factors included mouldy straw and incorrect management.

Animals↗
PubMed2005

Does high organochlorine (OC) exposure impair the resistance to infection in polar bears (Ursus maritimus)? Part II: Possible effect of OCs on mitogen- and antigen-induced lymphocyte proliferation.

Previous studies have reported alarmingly high levels of organochlorines (OCs), particularly polychlorinated biphenyls (PCBs), in free-ranging polar bears (Ursus maritimus). In this study plasma concentration of PCBs ranged from 14.8 to 200 ng/g wet weight. The aim of the study was to investigate associations between OCs and lymphocyte proliferation after in vitro stimulation with different mitogens and antigens. In 1998 and 1999, 26 and 30 free-ranging polar bears from Svalbard and Churchill, Canada, respectively, were recaptured 32-40 d following immunization with inactivated tetanus toxoid and hemocyanin from keyhole limpets (KLH) to sensitize lymphocytes. At recapture, blood was sampled for determination of plasma levels of PCBs and organochlorine pesticides (OCPs) and lymphocyte proliferation after in vitro stimulation with specific mitogens--phytohemagglutinin (PHA), pokeweed mitogen (PWM), concanavalin A (Con A), lipopolysaccharide (LPS), and purified protein derivative of Mycobacterium avium subsp. paratuberculosis (PPD)--and antigens: tetanus toxoid and KLH. The combinations of sum(PCBs) (sum of 12 individual PCB congeners), sum(OCPs) (sum of 6 OCPs), and their interactions contributed up to 15% of the variations in the lymphocyte responses. By using multiple regression analyses, followed by classical mathematic function analyses, thresholds for immunomodulation were estimated. Depending on the lymphocyte proliferation response studied, the estimated thresholds for significant immunomodulation were within the concentration ranges 32-89 ng/g wet weight (ww) and 7.8-14 ng/g ww for sum(PCBs) and sum(OCPs), respectively. Thus, this study demonstrated that OC exposure significantly influences specific lymphocyte proliferation responses and part of the cell-mediated immunity, which also is associated with impaired ability to produce antibodies (Lie et al., 2004).

Animals↗
PubMed1994

Purification, characterization, gene sequence, and significance of a bacterioferritin from Mycobacterium leprae.

The study of tissue-derived Mycobacterium leprae provides insights to the immunopathology of leprosy and helps identify broad molecular features necessary for mycobacterial parasitism. A major membrane protein (MMP-II) of in vivo-derived M. leprae previously recognized (Hunter, S.W., B. Rivoire, V. Mehra, B.R. Bloom, and P.J. Brennan. 1990. J. Biol. Chem. 265:14065) was purified from extracts of the organism and partial amino acid sequence obtained. This information allowed recognition, within one of the cosmids that encompass the entire M. leprae genome, of a complete gene, bfr, encoding a protein of subunit size 18.2 kD. The amino acid sequence deduced from the major membrane protein II (MMP-II) gene revealed considerable homology to several bacterioferritins. Analysis of the native protein demonstrated the iron content, absorption spectrum, and large native molecular mass (380 kD) of several known bacterioferritins. The ferroxidase-center residues typical of ferritins were conserved in the M. leprae product. Oligonucleotides derived from the amino acid sequence of M. leprae bacterioferritin enabled amplification of much of the MMP-II gene and the detection of homologous sequences in Mycobacterium paratuberculosis, Mycobacterium avium, Mycobacterium tuberculosis, Mycobacterium intracellulare, and Mycobacterium scrofulaceum. The role of this iron-rich protein in the virulence of M. leprae is discussed.

Amino Acid Sequence↗
PubMed2003

Targeting enteric bacteria in treatment of inflammatory bowel diseases: why, how, and when.

PURPOSE: This review discusses the role of bacterial adjuvants and antigens in induction and reactivation of chronic intestinal inflammation in susceptible hosts; discusses the results of recent therapeutic trials of antibiotics, probiotics, and prebiotics; and suggests future treatment strategies. RECENT FINDINGS: Bacterial adjuvants, including peptidoglycan, lipopolysaccharide, and DNA (CpG) bind to membrane-bound toll-like receptors (TLR-2, 4, and 9. respectively) or cytoplasmic (NOD1 and NOD2) receptors (pattern recognition receptors) that activate nuclear factor-kappaB and transcription of many proinflammatory cytokines and adhesion, costimulatory, and major histocompatibility complex class II molecules. Experimental enterocolitis does not occur in a sterile (germ-free) environment and is prevented and treated by broad-spectrum antibiotics. Individual nonpathogenic intestinal bacterial species selectively induce experimental colitis, with host specificity. Crohn disease and ulcerative colitis patients exhibit pathogenic immune responses (loss of immunologic tolerance) to multiple normal enteric bacterial species and serologic responses to Mycobacterium paratuberculosis. Metronidazole and ciprofloxacin selectively treat colonic Crohn disease, but not ulcerative colitis or ileal Crohn disease, and may prevent recurrence of postoperative Crohn disease. Certain probiotic species decrease relapse of ulcerative colitis and chronic pouchitis and delay onset of pouchitis. SUMMARY: Normal, nonpathogenic enteric bacteria induce and perpetuate chronic intestinal inflammation in genetically susceptible hosts with defective immunoregulation, bacterial clearance, or mucosal barrier function. Altering the composition and decreasing mucosal adherence/invasion of commensal bacteria with antibiotics, probiotics, and prebiotics can potentially prevent and treat Crohn disease, pouchitis, and possibly ulcerative colitis, but optimal treatments have not yet been identified.

Journal Article↗
PubMed2006

Mycobacteria in Crohn's disease: a persistent hypothesis.

Efforts to explore a mycobacterial origin for Crohn's disease typically have involved an epidemiological approach, searching for Mycobacterium avium subsp. paratuberculosis in patient tissue. An alternative approach involves consideration of genetic and experimental data regarding host resistance to mycobacteria. From human and mycobacterial genetics, it is known that mycobacterial diseases depend on both pathogen and host factors and that tuberculosis and leprosy are effectively genetic diseases. The discovery of a number of Crohn's susceptibility genes, including NOD2/CARD15, demonstrates that Crohn's also is a complex genetic disease. Mutations in NOD2/CARD15 do not necessarily lead to Crohn's disease, so other mitigating factors, genetic and/or environmental, probably are required to produce illness. Recent work has shown that NOD2/CARD15 serves a role in bacterial sensing and activation of innate immune responses, providing a link between Crohn's genetics and an environmental factor, potentially a bacterial trigger. In this review, we discuss the current understanding of mycobacterial and Crohn's genetic susceptibility and review the evidence that NOD2/CARD15 may mediate host resistance to mycobacterial infection.

Animals↗
PubMed1990

Towards a phylogeny and definition of species at the molecular level within the genus Mycobacterium.

16S rRNA sequences from Mycobacterium tuberculosis, M. avium, M. gastri, M. kansasii, M. marinum, M. chelonae, M. smegmatis, M. terrae, M. gordonae, M. scrofulaceum, M. szulgai, M. intracellulare, M. nonchromogenicum, M. xenopi, M. malmoense, M. simiae, M. flavescens, M. fortuitum, and M. paratuberculosis were determined and compared. The sequence data were used to infer a phylogenetic tree, which provided the basis for a systematic phylogenetic analysis of the genus Mycobacterium. The groups of slow- and fast-growing mycobacteria could be differentiated as distinct entities. We found that M. simiae occupies phylogenetically an intermediate position between these two groups. The phylogenetic relatedness within the slow-growing species did not reflect the Runyon classification of photochromogenic, scotchromogenic, and nonchromogenic mycobacteria. In general, the phylogenetic units identified by using rRNA sequences confirmed the validity of phenotypically defined species; an exception was M. gastri, which was indistinguishable from M. kansasii when this kind of analysis was used.

Base Sequence↗
PubMed1994

Nucleotide sequence comparison of the mycobacterial dnaJ gene and PCR-restriction fragment length polymorphism analysis for identification of mycobacterial species.

We recently reported a genus-specific PCR for the mycobacterial dnaJ gene. In the present study, we have determined the nucleotide sequences of the dnaJ gene from 19 mycobacterial species (Mycobacterium tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. microti, M. marinum, M. kansasii, M. gastri, M. simiae, M. scrofulaceum, M. szulgai, M. gordonae, M. avium, M. intracellulare, M. xenopi, M. fortuitum, M. chelonae, M. hemophilum, and M. paratuberculosis). On the basis of the amplified dnaJ gene nucleotide sequences, we constructed a phylogenetic tree of the mycobacterial species by using the neighbor-joining method and unweighted pairwise grouping method of arithmetic average. We found that the phylogenetic relationship inferred within the slowly growing species was in good agreement with the traditional classification, with three major branches corresponding to Runyon's groups I, II, and III. An exception was M. simiae, which was phylogenetically closer to the cluster including members of Runyon's group III than to that of Runyon's group I. On the other hand, the rapid growers, such as M. fortuitum and M. chelonae, did not form a coherent line corresponding to Runyon's group IV, indicating that our phylogenetic analysis based on the dnaJ gene reflects the phenotypic characteristics such as pigmentation but not the growth rate. Finally, we revealed the species-specific restriction sites within the amplified dnaJ gene to differentiate most of the mycobacterial DNA by a combination of PCR with restriction fragment length polymorphism analysis.

Amino Acid Sequence↗
PubMed1990

Discovery of an insertion sequence, IS116, from Streptomyces clavuligerus and its relatedness to other transposable elements from actinomycetes.

We have identified an insertion sequence, IS116, present in Streptomyces clavuligerus at one copy per genome. The element was discovered as a 1.4 kb insertion into the multicopy plasmid pIJ702 after propagation in S. clavuligerus. The nucleotide sequence of IS116 and the flanking sequences from pIJ702 have been determined. The junctions with pIJ702 show no target site duplication and there are no inverted repeats at the ends of the element. One putative coding open reading frame of 1197 bp was identified which would code for a protein product of 399 amino acids. This protein resembles deduced integrase/transposase proteins specified by three other transposable elements of actinomycetes: IS110 and the mini-circle from Streptomyces coelicolor A3(2), and--most particularly--IS900 of Mycobacterium paratuberculosis. Two regions that are relatively conserved among these gene products show features found in similar positions in many reverse transcriptases. IS116 and IS900 are also closely similar in their general organization and (apparently) in their insertion site specificity, whereas IS110 and the mini-circle are quite different in these features.

Actinomycetales↗
PubMed1992

IS902, an insertion element of the chronic-enteritis-causing Mycobacterium avium subsp. silvaticum.

An insertion sequence element of Mycobacterium avium subsp. silvaticum was isolated and its complete nucleotide sequence determined. IS902 is 1470 bp in size and is repeated 10-12 times per genome. An open reading frame of 1200 bp was identified, encoding a protein product of Mr 43932. This protein is highly similar to the predicted proteins of IS900 of Mycobacterium paratuberculosis, IS116 of Streptomyces clavuligerus and IS110 of Streptomyces coelicolor. IS902 lacks terminal inverted repeats and flanking direct repeats but displays insertion site specificity.

Amino Acid Sequence↗
PubMed1993

Nucleic acid sequence-based amplification (NASBA) for the identification of mycobacteria.

Nucleic acid sequence-based amplification (NASBA), an isothermal amplification technique for nucleic acids (NA), was investigated for the species-specific identification of mycobacteria. A set of primers was selected from a highly conserved region of the 16S rRNA sequence of mycobacteria sandwiching a variable sequence to perform amplification of mycobacterial RNA. Species-specific probes for the M. tuberculosis complex, M. avium-paratuberculosis, M. intracellulare and M. leprae were hybridized in-solution with the amplified nucleic acids of 10 pathogenic mycobacteria and 11 closely related bacteria, as well as with human-derived NA in an enzyme-linked gel assay (ELGA). Each probe was shown to hybridize specifically to the amplified single-stranded RNA of the corresponding species. Thirty-two clinical isolates of M. tuberculosis strains from different parts of the world were correctly identified by NASBA using the M. tuberculosis-complex-specific probe. In combination with the ELGA, NASBA could identify mycobacteria rapidly, i.e. in less than 6 h. The relative simplicity and rapidity of this technique makes it an attractive tool for species-specific identification of mycobacteria.

Base Sequence↗
PubMed1999

IS1626, a new IS900-related Mycobacterium avium insertion sequence.

An insertion sequence designated IS1626 was isolated and characterized from a Mycobacterium avium clinical strain. IS1626 was detected by high-stringency hybridization with the pMB22/S12 probe from IS900 of Mycobacterium paratuberculosis. IS1626 is 1418 bp in size and has a G+C content of 65 mol%. It has neither terminal inverted repeats nor flanking direct repeats. Analysis of three IS1626 insertion sites in the M. avium strain and the corresponding potential insertion sites in two IS1626-free M. avium strains indicated a consensus sequence of CATGCN(4-5)TCCTN(2)G for IS1626 insertion. In the three clones examined, IS1626 has the same orientation with respect to this target site. IS1626 has two major ORFs. ORF1179 encodes a predicted protein of 393 amino acids. ORF930, on the complementary strand of ORF1179, encodes a protein of 310 amino acids. The Shine-Dalgarno sequence for ORF930 is partially located in the flanking region, similar to other IS900-related elements. Analysis of the comparable features of insertion sequences and their variable occurrence in related organisms is useful for studying the evolution of these elements and their hosts.

Amino Acid Sequence↗
PubMed1999

The Mycobacterium tuberculosis katG promoter region contains a novel upstream activator.

An Escherichia coli-mycobacterial shuttle vector, pJCluc, containing a luciferase reporter gene, was constructed and used to analyse the Mycobacterium tuberculosis katG promoter. A 1.9 kb region immediately upstream of katG promoted expression of the luciferase gene in E. coli and Mycobacterium smegmatis. A smaller promoter fragment (559 bp) promoted expression with equal efficiency, and was used in all further studies. Two transcription start sites were mapped by primer extension analysis to 47 and 56 bp upstream of the GTG initiation codon. Putative promoters associated with these show similarity to previously identified mycobacterial promoters. Deletions in the promoter fragment, introduced with BAL-31 nuclease and restriction endonucleases, revealed that a region between 559 and 448 bp upstream of the translation initiation codon, designated the upstream activator region (UAR), is essential for promoter activity in E. coli, and is required for optimal activity in M. smegmatis. The katG UAR was also able to increase expression from the Mycobacterium paratuberculosis P(AN) promoter 15-fold in E. coli and 12-fold in M. smegmatis. An alternative promoter is active in deletion constructs in which either the UAR or the katG promoters identified here are absent. Expression from the katG promoter peaks during late exponential phase, and declines during stationary phase. The promoter is induced by ascorbic acid, and is repressed by oxygen limitation and growth at elevated temperatures. The promoter constructs exhibited similar activities in Mycobacterium bovis BCG as they did in M. smegmatis.

Bacterial Proteins↗
PubMed2001

Unique expression of a highly conserved mycobacterial gene in IS901(+) Mycobacterium avium.

Expression of a gene encoding a novel protein antigen of 40 kDa (p40) was detected in IS901(+) strains of Mycobacterium avium, but not in any other species or subspecies of Mycobacterium tested, including IS901(-) M. avium and the other members of the M. avium complex. Although Southern hybridization revealed that the p40 gene is widely distributed within the genus, expression of the antigen could not be detected on Western blots of mycobacterial cell lysates. Nucleotide sequence analysis of the cloned p40 gene, and a database search, revealed high levels of sequence identity with a homologous gene in IS901(-) M. avium, M. avium subsp. paratuberculosis, Mycobacterium bovis, Mycobacterium leprae, Mycobacterium smegmatis and Mycobacterium tuberculosis. Further analysis of upstream sequences identified a putative promoter region. The p40 gene is the first example of a gene that is widely distributed within the genus Mycobacterium but expressed only in association with the presence of a genomic insertion element, in this case IS901, in strains of M. avium isolated from birds and domestic livestock.

Animals↗
PubMed1978

The dependence of some strains of Mycobacterium avium on mycobactin for initial and subsequent growth.

This paper demonstrates that various degrees of mycobactin dependence exist within the M. avium group of mycobacteria and that this characteristic is not confined to M. paratuberculosis. The strains employed came from a wide range of animal species and all were virulent for chickens. Mycobactin dependence was observed in the M. avium serotypes 1, 2 and 3. The importance of employing a medium containing mycobactin for primary isolations observation may be of interest in relation to the primary isolation of other species of mycobacteria.

Animals↗