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PubMed1990

Characterization of Mycobacterium paratuberculosis and "wood-pigeon" mycobacteria by isoenzyme profile and selective staining of immunoprecipitates.

Cell-free extracts of various strains belonging to Mycobacterium paratuberculosis (Ptb) and "wood-pigeon" (WP) mycobacteria were compared by polyacrylamide gel electrophoresis and the various protein bands obtained were tested for peroxidase enzyme activity. One strain of Mycobacterium avium served as a control. Bacterial extracts were also analysed by crossed immunoelectrophoresis (CRIEP) and fused rocket immunoelectrophoresis (FRIEP) using antisera raised in rabbit against M. paratuberculosis and WP mycobacteria. The immunoprecipitates obtained both in CRIEP and FRIEP plates were subsequently stained for selective peroxidase enzyme staining. Our results showed that, although Ptb and WP mycobacteria shared common peroxidase isoenzymes and antigens, they also had specific immunoprecipitates showing the differences between the two groups of bacteria.

Electrophoresis, Polyacrylamide Gel↗
PubMed1989

Sequence and characteristics of IS900, an insertion element identified in a human Crohn's disease isolate of Mycobacterium paratuberculosis.

The complete sequence of an insertion element IS900 in Mycobacterium paratuberculosis is reported. This is the first characterised example of a mycobacterial insertion element. IS900 consists of 1451bp of which 66% is G + C. It lacks terminal inverted and direct repeats, characteristic of Escherichia coli insertion elements but shows a degree of target sequence specificity. A single open reading frame (ORF 1197) coding for 399 amino acids is predicted. This amino acid sequence, and to a lesser extent the nucleotide sequence, show significant homologies to IS110, an insertion element of Streptomyces coelicolor A3(2). It is proposed that IS900, IS110, and similar insertion elements recently identified in disease isolates of Mycobacterium avium are members of a phylogenetically related family. IS900 will provide highly specific markers for the precise identification of Mycobacterium paratuberculosis, useful in defining its relationship to animal and human diseases.

Amino Acid Sequence↗
PubMed1994

Identification and characterization of a putative serine protease expressed in vivo by Mycobacterium avium subsp. paratuberculosis.

A putative serine protease expressed in vivo by Mycobacterium avium subsp. paratuberculosis was isolated from a lambda gt11 genomic expression library by screening with serum from a naturally infected sheep. The gene was contained in two overlapping clones, which were shown by antibody elution to encode a protein of 34 kDa in M. a. paratuberculosis. The clones were sequenced and database searches detected a motif identical to the active serine site in trypsin, and 30% homology to the putative serine proteases (HtrA proteins) of Escherichia coli, Salmonella typhimurium, Brucella abortus and Rochalimaea henselae.

Amino Acid Sequence↗
PubMed1994

The effect of vaccination on the prevalence of paratuberculosis in large dairy herds.

Eight hundred and sixty-six one-month-old female calves were vaccinated using heat-killed Mycobacterium paratuberculosis in oil during a five-year period. Seven hundred and twenty-one bull calves and 379 female calves served as nonvaccinated controls. Two large herds were used in the study. The calves were tested by faecal culture and by serologic tests on the day of vaccination, at 3, 6, 9 and 12 months of age, at breeding and on the day of parturition. The results were evaluated by trend analyses. Following vaccination, the faecal shedding of bacteria was greatly reduced as determined by annual faecal microscopic tests. During the last 6 months of the experiment as few as 9 samples from 612 cattle proved positive by microscopy and/or culture. The number of seropositive animals and the antibody titres (CFT and AGID) increased during the first three years, then declined.

Animals↗
PubMed1992

Protein antigens secreted by Mycobacterium paratuberculosis.

Proteins secreted by Mycobacterium paratuberculosis (M.ptb) during short-term cultivations were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western(Immuno) blotting. Cultivation in a defined medium containing 35S methionine allowed autoradiographic detection of proteins which had been secreted or passively released by actively metabolizing M.ptb organisms. After the first 3 days of cultivation, 4 proteins with molecular weights of approximately 38, 50, 65 and 110 kilodaltons (kd) were detected on SDS gels. Longer incubation up to 12 days resulted in an increased concentration of these proteins as well as in appearance of additional proteins ranging from 14 to over 90 kd. In long-term (8-10 weeks) culture filtrates only two prominent proteins with molecular weights of 30 and 65 kd proteins could be detected. Immunoblot analysis showed that some of the proteins secreted during short-term cultivations were recognized by sera from M.ptb-infected sheep and more significantly by sera from animals which had been immunized with a M.ptb live vaccine strain. The study indicates that during short incubation times M.ptb may secrete immunoreactive proteins which are not dominant in long-term cultures.

Animals↗
PubMed1993

Comparative analysis of three sensitins used in cutaneous testing for tuberculosis and paratuberculosis in cattle.

Correspondence between components of sensitins (avian, bovine, and johnin-PPD tuberculins) and the proteins of the corresponding microorganisms (Mycobacterium avium, Mycobacterium bovis and Mycobacterium paratuberculosis) was established. Most of these identified proteins are present in the three organisms, but the 38.3- and 48.8-kDa proteins of M. avium and the 54.0- and 58.7-kDa proteins of M. bovis are endowed with species-specific epitope(s).

Animals↗
PubMed1993

Reproducibility of a commercial enzyme-linked immunosorbent assay for bovine paratuberculosis among eight laboratories.

Interlaboratory reproducibility of an absorbed enzyme-linked immunosorbent assay (ELISA) kit for detection of bovine serum antibodies to Mycobacterium paratuberculosis was evaluated. A panel of 30 bovine sera (15 positives and 15 negatives) was tested in triplicate microtiter wells on each of 2 days at 8 different laboratories. One laboratory had invalid results because of positive or negative serum control optical density (OD) readings beyond the acceptable range specified by the kit. The coefficient of variation (CV) for mean OD values was influenced by low ODs on test negative sera at 2 laboratories, thus the CVs on positive sera were considered a more representative measure of kit reproducibility. Between-well CVs averaged 6.7% +/- 2.8% (mean +/- standard deviation), and between-day CVs averaged 14.5% +/- 9.8% among the 7 laboratories with valid assays on the 15 positive sera. The OD values were converted to positive or negative classifications for each assay well, and the results were compared. Among 1,392 assays in 7 laboratories, 98.6% were in agreement. Eleven of 18 discrepant results were due to a sample that consistently gave OD values near the cutoff for a positive test. Exclusion of that serum from the analysis resulted in a 99.8% rate of agreement among laboratories. Results indicated that the absorbed ELISA kit provided reproducible results within and between laboratories.

Analysis of Variance↗
PubMed1992

Paratuberculosis vaccine in a large dairy herd.

On a 500-cow dairy farm a total of 866 young calves less than one month old were vaccinated with a heat-killed oil-adjuvated bacterin against Mycobacterium paratuberculosis over a period of five years. The vaccinated calves were tested by faecal microscopy, bacteriology and serology on the day of vaccination, at the age of 3, 6, 9 and 12 months, at breeding age, and on the day of calving. A total of 721 bull calves and 379 female calves served as unvaccinated controls in two groups. The results were evaluated by trend analyses. Vaccination greatly reduced the faecal shedding of mycobacteria as demonstrated by the annual faecal microscopic examinations. During the last 6 months of the experiment only 9 of 612 samples were found positive by microscopy and by bacterial culture. The number of seropositive animals and the antibody titres demonstrated by the complement fixation test (CFT) and agar gel immunodiffusion (AGID) increased during the first three years. Later on, both the number of seropositive animals and CFT titres decreased.

Animals↗
PubMed1995

Solid-phase hybridization capture of low-abundance target DNA sequences: application to the polymerase chain reaction detection of Mycobacterium paratuberculosis and Mycobacterium avium subsp. silvaticum.

Polymerase chain reaction (PCR) has been widely applied to the detection of microorganisms. Overall sensitivity of PCR tests may be substantially reduced due to a large excess of nontarget DNA and inhibitory substances in the sample. We used a 5'-biotinylated 513-bp probe from the 3' region of the IS 900 element specific for Mycobacterium paratuberculosis (Mptb) to capture target Mptb DNA from crude sample DNA extracts. Captured target DNA was separated using streptavidin-coated magnetic particles (Dynal). Since the IS 900 element shares homology over this region with IS 902 in Mycobacterium avium subsp. silvaticum (Mavs), target DNA from this other pathogen was also retained. Highly specific PCR for the detection of either organism directed to the 5' regions of IS 900 or IS 902 was then performed directly on the solid phase. Hybridization capture of target DNA using sequence adjacent to the desired specific PCR site applied to Mptb increased overall sensitivity of detection in tissue and fecal extracts 10- to 100-fold. False positives due to contamination artifact were substantially excluded since the capture probe did not retain amplicons from the detection PCR. Development of the method to involve covalent 5' immobilization of capture probes on heat-resistant polymers should, in the future, provide a simple system with broad potential applications.

Animals↗
PubMed1991

Antibody reactivities of Mycobacterium paratuberculosis infected sheep as analyzed by enzyme-linked immunosorbent assay and western blotting.

Antibody reactivities in sera from Mycobacterium paratuberculosis (M. ptb) infected and vaccinated sheep were analyzed by enzyme-linked immunosorbent assay (ELISA) and Western (immuno)blotting using a sonicate antigen from M. ptb. Both methods allowed good differentiation between infected/vaccinated animals and noninfected controls. Removal of nonspecific crossreactive antibodies by absorption with a M. phlei sonicate antigen coupled to Sepharose reduced ELISA reactivities of positive sera by 50% and those of noninfected serum by 85%. Immunoblotting analysis revealed that reduction by M. phlei absorption was due to lower reactivities of M. ptb antigens in the range of 30 to 45 kDa. However, one protein with a molecular mass of approx. 27 kDa seemed to be specific for M. ptb since it reacted similarly with nonabsorbed and absorbed serum but not with antibodies which were eluted from M. phlei-Sepharose after absorption. Our findings indicate that M. ptb and M. phlei share a number of common antigens of potential pathogenic importance and that only a smaller part of proteins (i.e. the 27 kDa protein) might be specific for M. ptb.

Animals↗
PubMed1992

Molecular cloning and characterization of Mycobacterium paratuberculosis promoters in Escherichia coli.

DNA fragments from Mycobacterium paratuberculosis were cloned in the promoter probe plasmid pKO1. Of 957 recombinant DNA clones, 24 induced synthesis of galactokinase (the reporter gene) when these plasmids were transformed into an Escherichia coli strain deficient for the enzyme. A DNA insert from one putative promoter-containing plasmid, designated pAG5, was sequenced and shown to contain, a characteristic RNA polymerase binding site, a probable ribosomal binding site and a putative open reading frame.

Amino Acid Sequence↗
PubMed1990

Immunohistochemical study of bovine lymph nodes with antibodies against S100 protein subunits: comparison between lymph nodes of healthy and Mycobacterium paratuberculosis-infected cattle.

Using immunohistochemistry, the differential distribution of the alpha subunit (S100 alpha) and beta subunit (S100 beta) of S100 protein was studied in mesenteric lymph nodes from normal or Mycobacterium paratuberculosis-infected cattle. In epithelioid cell granulomas, S100 alpha-positive epithelioid cells and some giant cells were scattered among S100 alpha-negative cells, which were predominant. The S100 beta-positive and -negative cells contained acid-fast bacilli. The presence of S100 beta-positive cells was not demonstrated in the granulomas. In normal component cells in the lymph nodes, follicular dendritic cells in the germinal centres and endothelium of lymphatic sinus and lymph vessels were positive for S100 alpha. S100 beta was positive only in the endothelial cells of blood vessels. Results shown in the present paper are discussed in light of results obtained in other work on human tissues using the same sources of antibodies.

Animals↗
PubMed1992

Mycobacterium paratuberculosis binds fibronectin.

Fibronectin, an adhesive glycoprotein which is present in plasma and on many host cell surfaces of many host organisms, binds to certain bacterial pathogens. This study demonstrates the ability of Mycobacterium paratuberculosis (M.ptb) to interact with 125I-labelled fibronectin purified from bovine and ovine plasma. Two M.ptb strains were tested: a clinical isolate and a commercially available vaccine strain. Both strains showed significant fibronectin-binding activities of 22 and 41%, respectively, whereas non-pathogenic M.phlei had almost no affinity for fibronectin. Binding activities were similar for ovine and bovine fibronectin. We found that fibronectin binding by M.ptb was (1) time-dependent, reaching saturation within 90 min, (2) specific, since it was inhibited by an excess of unlabelled fibronectin but not by albumin, (3) saturable, with an apparent dissociation constant of 1.25 x 10(-9) M and a maximal number of 1,600 binding sites per bacterium, and (4) sensitive to detergents, proteases and heat treatments, indicating the protein nature of the responsible binding component(s). Scatchard plot analysis gave a straight line suggesting the presence of a single type of fibronectin receptor on M.ptb.

Animals↗
PubMed2026

Matching genomic evidence to claims about Mycobacterium avium subsp. paratuberculosis: Host association, host adaptation, mechanism, and virulence.

Whole-genome sequencing (WGS) permits high-resolution comparison of Mycobacterium avium subsp. paratuberculosis (MAP) isolates and pangenome analysis. Combined with animal-movement data, WGS can support transmission inference, but resolution alone does not establish the biological meaning of genomic variation. This focused narrative review applies a two-dimensional framework to purposively selected MAP studies, separating claim targets from support profiles. Claim targets include lineage identity, host-source or lineage characterization, host association, transmission, candidate genomic features, measured bacterial or host-cell phenotypes, natural-host infection fitness, disease or damage, shedding, and control outcomes. Depending on the claim, evidence operations may include characterization, context-aware comparative inference, direct endpoint ascertainment, and controlled feature perturbation; these are non-ordinal and may co-occur. On-target attribution, independent replication, and transportability are reported separately. The claim, not the study, is the unit of assessment. Typing markers support isolate or lineage discrimination, whereas phylogenomics supports evolutionary inference; neither alone establishes host adaptation. Pangenome comparisons and microbial genome-wide association studies nominate candidate features rather than establish adaptation. Cell-envelope and iron-associated studies support specified biochemical, transcriptional, or physiological phenotypes under defined conditions, while macrophage and calf models support only the endpoints measured. Annotated sequence variation alone nominates pathogenicity hypotheses. Across the illustrative studies selected here, MAP genomics most directly supported lineage classification, candidate discovery, measured bacterial phenotypes, bounded transmission inference, and natural-host infection-fitness claims. Claims about adaptation, mechanism, virulence, or control require endpoints and comparisons matched to the stated claim and model; feature-specific causal claims additionally require evidence linking the bacterial feature to the measured endpoint.

Claim boundaries↗
PubMed1986

Participation of iron on the growth inhibition of pathogenic strains of mycobacterium avium and M. paratuberculosis in serum.

Serum, which is low in freely-available iron, was bacteriostatic for M. avium and M. paratuberculosis irrespective of the ability of these organisms to produce the iron-chelators mycobactin and exochelin for iron acquisition. Bacteriostasis was overcome when the serum was supplemented with exochelin or mycobactin, but only if the mycobacteria had previously adapted to growth in a low iron environment. The results imply that for studies on mycobacterial pathogenicity the test cultures should be grown under low iron conditions before being used. The role of the exochelins, mycobactin and iron in the development of in vivo growth is discussed.

Animals↗
PubMed1991

Antimicrobial activity of rifabutin in combination with two and three other antimicrobial agents against strains of Mycobacterium paratuberculosis.

The inhibitory and bactericidal, synergistic, and antagonistic activities of rifabutin combined with ciprofloxacin, ethambutol, clofazimine, cefazolin, and amikacin in dual and triple combinations against various human and animal isolates of Mycobacterium paratuberculosis were determined. Synergism was observed when rifabutin was combined with either cefazolin or clofazimine in double combinations. The greatest amount of synergy occurred with the rifabutin-cefazolin combination in which bactericidal synergism was present with all strains. Of the triple combinations examined, only rifabutin in combination with ethambutol and cefazolin or streptomycin and cefazolin showed bactericidal synergism against most of the strains. Although antagonism was not observed in any double combination with rifabutin, antagonism was shown with several of the triple combinations. The rifabutin-cefazolin and rifabutin-streptomycin-cefazolin combinations were found to have the greatest bactericidal synergism at concentrations well within achievable serum and tissue levels and may be appropriate choices for chemotherapeutic use.

4-Quinolones↗
PubMed1992

p43, the protein product of the atypical insertion sequence IS900, is expressed in Mycobacterium paratuberculosis.

The novel mycobacterial insertion sequence IS900 was analysed by coupled transcription-translation, of both strands independently, in a cell-free E. coli extract using an exogenous promoter. This revealed only one protein product, p43, as predicted from the nucleotide sequence. The protein was readily translated in recombinant E. coli, using the tac promoter, though it did not appear as a major product by SDS-PAGE analysis. A synthetic peptide was used to generate and affinity-purify a specific anti-p43 antibody, which clearly identified the protein in recombinant E. coli. p43 was relatively stable in exponential phase and stationery phase bacteria, though a 28 kDa processed form was seen to accumulate over a period of hours. Both forms appeared in the soluble fraction of the bacterial lysate. The anti-p43 antibody also identified p43, as a 28 kDa processed product, in Western blots of protein extracts from Mycobacterium paratuberculosis, indicating a level of expression which would be unusually high for a classical transposase. These data have important implications for the relationship between IS900 and its host.

Amino Acid Sequence↗
PubMed1992

Expression of Escherichia coli beta-galactosidase in Mycobacterium bovis BCG using an expression system isolated from Mycobacterium paratuberculosis which induced humoral and cellular immune responses.

A promoter sequence, PAN, was isolated from Mycobacterium paratuberculosis and characterized. This promoter lies adjacent to, and outside, the 3' end of an IS900 insertion element. IS900 contains an open reading frame, ORF2, on the complementary strand which codes for the putative transposase of this insertion sequence. A DNA fragment containing PAN and part of ORF2 was fused to the lacZ gene and inserted into the replicative shuttle vector pRR3. Mycobacterium smegmatis and Mycobacterium bovis BCG (BCG) transformed with this plasmid exhibited beta-galactosidase activity. However, lacZ was only expressed in Escherichia coli under the control of PAN, when ORF2 was deleted. Immunization of mice with the recombinant M. bovis BCG expressing lacZ resulted in the induction of a high humoral and cellular response directed against beta-galactosidase. The PAN-ORF2 expression system may prove to be particularly useful for cloning and expression of heterologous genes in the BCG vaccine strain.

Amino Acid Sequence↗