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PubMed1980

Bovine paratuberculosis I. A herd study using complement fixation and intradermal tests.

A dairy herd (102 cattle) which had been enrolled under a paratuberculosis control program for two years utilizing a complement fixation test (carbohydrate antigen) and intradermal skin test (johnin PPD) was subjected to two further herd tests and followed to slaughter to determine infection status by culture and histology. Mycobacterium paratuberculosis infection was demonstrated in 37 of the animals of which only five were considered reactors on the basis of the last two herd tests applied. Cultural and histopathological evaluation indicated the testing procedures had eliminated heavily infected animals. The limitations of these testing procedures under free stall housing conditions are discussed.

Animals↗
PubMed1980

Bovine paratuberculosis II. A comparison of fecal culture and the antibody response.

Fecal culture for Mycobacterium paratuberculosis and a complement fixing serological test using a carbohydrate antigen were compared for diagnostic efficiency in cattle naturally infected with M. paratuberculosis. Serological reactivity was associated with the persistent fecal shedding of large numbers of bacteria and in the absence of false positives in the population studied, was considered an efficient method for the identification of this segment of infected cattle. Minimally infected cattle, which inconsistently shed small numbers of bacteria, were generally serologically negative. One persistent fecal shedder demonstrated a fluctuating serological response to the carbohydrate antigen following abortion. The advantages and disadvantages of this serological diagnostic procedure in relationship to fecal culture are discussed.

Animals↗
PubMed1977

Distribution of paratuberculosis in Wisconsin, by soil regions.

A study was undertaken to determine the relationship between concentration of paratuberculosis-infected herds and soil types in Wisconsin. Locations of farms on which paratuberculosis had occurred between 1971 and 1975 were obtained from the Wisconsin Department of Agriculture. The disease occurred mainly in the rolling and undulating areas. Fifty percent of the infection herds were in the southwestern "driftless area" and the southeast uplands; 43% occurred in the western part of the northern silty and loamy uplands; the other 7% were scattered in the east and east-central part of the state. The disease did not necessarily occur in the areas of high cattle concentration. It was concluded that the disease persisted in regions with acid soils but not in regions with alkaline, calcereous soils.

Animals↗
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↗
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↗
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↗
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↗
PubMed1990

Immunohistochemical methods for the visualization of Mycobacterium paratuberculosis in bovine tissues.

The peroxidase-antiperoxidase (PAP), streptavidin-biotin (SB), and avidin-biotin-complex (ABC) techniques have been evaluated for the visualization of Mycobacterium paratuberculosis (Mp) in formalin-fixed, paraffin-embedded bovine tissues. The used immunoperoxidase techniques were comparatively better than the Ziehl-Neelsen stain, specially for the demonstration of small number of mycobacteria in tissue sections.

Animals↗