Specificity of the 34-kilodalton immunodominant protein of Mycobacterium avium sp. paratuberculosis.
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Amino Acid Sequence↗
Biomedical subjects
P Gilot
Publications and source records attributed to P Gilot.
Typing of Listeria monocytogenes strains by repetitive element sequence-based PCR.
Listeria monocytogenes strains possess short repetitive extragenic palindromic (REP) elements and enterobacterial repetitive intergenic consensus (ERIC) sequences. We used repetitive element sequence-based PCR (rep-PCR) to evaluate the potential of REP and ERIC elements for typing L. monocytogenes strains isolated from humans, animals, and foods. On the basis of rep-PCR fingerprints, L. monocytogenes strains were divided into four major clusters matching origin of isolation. rep-PCR fingerprints of human and animal isolates were different from those of food isolates. Computer evaluation of rep-PCR fingerprints allowed discrimination among the tested serotypes 1/2a, 1/2b, 1/2c, 3b, and 4b within each major cluster. The index of discrimination calculated for 52 epidemiologically unrelated isolates of L. monocytogenes was 0.98 for REP- and ERIC-PCR. Our results suggest that rep-PCR can provide an alternative method for L. monocytogenes typing.
Animals↗
PCR assay fails to detect molluscum contagiosum virus-related sequences in AIDS-related Kaposi's sarcoma.
Previous PCR-based studies have demonstrated the presence of various viral DNA or RNA sequences in Kaposi's sarcoma (KS) tissues. To date, only human herpesvirus 8 (HHV-8) DNA sequences are found consistently in KS. The putative role of this agent in KS pathogenesis remains, however, to be determined; HHV-8 could infect populations endemically and could be reactivated in patients with KS. A close association between AIDS-related KS and molluscum contagiosum occurrence was found and this study was conducted primarily to search for the presence of molluscum contagiosum virus DNA sequences in KS. Frozen KS samples were examined for the presence of both HHV-8 and molluscum contagiosum virus DNA sequences by PCR. Despite a high rate of co-infection, no molluscum contagiosum virus (MCV) DNA sequence could be found in the KS samples whereas HHV-8 was uniformly detected. These results suggest that the high prevalence of MCV in AIDS patients with KS relies on a mode of transmission common for HHV-8 and molluscum contagiosum virus rather than on a multiviral etiology of KS. They may also indicate a particular susceptibility of the host to viral reactivation. If this is so, the failure to detect MCV DNA sequences in KS tissues by PCR indicates that locally produced or released cyotokines are not involved in the latter process.
AIDS-Related Opportunistic Infections↗
Sporadic case of listeriosis associated with the consumption of a Listeria monocytogenes-contaminated 'Camembert' cheese.
Listeria monocytogenes is an intracellular gram-positive organism responsible for severe infections in both humans and animals. Whereas the food-borne transmission of listeriosis was demonstrated in several outbreaks, most cases of listeriosis occur sporadically and are rarely linked with consumption of contaminated foods. In this paper a case of septicaemia with L. monocytogenes in a 73-year-old immunocompromised man is described. Evidence for the association of this case of listeriosis with the consumption of a contaminated 'Camembert' cheese is provided by serotyping, esterase typing, DNA macrorestriction patterns analysis and level of virulence of the isolated strains for mice.
Aged↗
Serotyping and esterase typing for analysis of Listeria monocytogenes populations recovered from foodstuffs and from human patients with listeriosis in Belgium.
Listeria monocytogenes strains isolated in Belgium from different foodstuffs and in sporadic cases of human listeriosis were analyzed. The distribution of serovars differed in each of these populations. The bacteria isolated from cheeses and from human patients with listeriosis were further studied by esterase typing. The twenty esterase patterns defined were not equally distributed in these two populations. The secretion of the virulence determinant phosphatidylinositol-specific phospholipase C and the pathogenicity level of strains in immunocompromised mice could not explain the unequal distribution of esterase types. The discrimination index of esterase typing (DI = 0.868) was compared with that of serotyping (DI = 0.666) and with that of the two combined methods (DI = 0.899).
Animals↗
Cloning and characterization of transcriptional promoters from Bacillus subtilis phage 2C.
Phage 2C is a Bacillus subtilis lytic phage, whose genome contains hydroxymethyluracil in place of thymine. To isolate promoters of early phage genes involved in the take-over of cellular metabolism, 2C DNA libraries were constructed in promoter-probe plasmids replicating in Escherichia coli and B. subtilis. Four different 2C DNA fragments strongly expressed reporter genes in E. coli but not in B. subtilis. All fragments originated from unique sequences of the genome and not from its terminal redundancies. One fragment was sequenced. Despite the presence of an sigma-A-RNA polymerase binding site upstream of the transcriptional initiation site of a 2C early gene, this fragment did not promote transcription in B. subtilis.
Bacillus Phages↗
Characterization of five esterases from Listeria monocytogenes and use of their electrophoretic polymorphism for strain typing.
Esterases from Listeria monocytogenes strains isolated from cheeses were analyzed by starch gel electrophoresis. Five esterases, numbered from EST 1 to EST 5 in order of decreasing anodal migration, were identified. The EST 1, EST 3, EST 4, and EST 5 set was most active toward alpha-naphthyl propionate, while EST 2 was most active toward alpha-naphthyl acetate. Results from inhibitor studies suggest that all of these esterases were EC class 3.1.1.1 carboxylesterases, except that EST 1 and EST 3 also showed some sensitivity to parahydroxymercuribenzoate. Polymorphism of these five esterases was observed in the population. Twelve esterase patterns were defined and used to subdivide serotypes.
Bacterial Typing Techniques↗
Mycobacterium paratuberculosis and Escherichia coli share common antigenic determinants.
Paratuberculosis (Johne's disease) is a chronic enteritis of ruminants, which is due to infection by Mycobacterium paratuberculosis. By comparative analysis of Western blots of M. paratuberculosis components incubated with sera from paratuberculous cows (either pre-absorbed or not on an Escherichia coli sonicate), we have shown the presence of cross-reactive antigens between M. paratuberculosis and E. coli. Components in the range of 22 to 75 kDa are recognized by sera from paratuberculous cows, but only some components in the range of 20 to 40 kDa are endowed with specific B-cell epitopes to M. paratuberculosis with respect to E. coli. Cross-reactions were further stressed by the fact that rabbit immunoglobulins to E. coli recognized some ten M. paratuberculosis components (in the range of 45 to 66 kDa). The importance of cross-reactivity between the two bacterial species was evaluated by enzyme-linked immunosorbent assay (ELISA) using soluble sonic-extract components from M. paratuberculosis as antigens. Pre-absorption of series of bovine sera with E. coli resulted in the diminution of ELISA mean optical density readings by 16.1% and 62.0% for paratuberculous or healthy animals, respectively.
Animals↗
Occurrence, in Crohn's disease, of antibodies directed against a species-specific recombinant polypeptide of Mycobacterium paratuberculosis.
Sera from patients with Crohn's disease and control were analyzed by an enzyme-linked immunosorbent assay based on the Mycobacterium paratuberculosis-specific recombinant polypeptide a362. Anti-a362 immunoglobulin G (IgG) (P < 0.05) and IgA (P < 0.001) titers were higher in patients with Crohn's disease than in controls. A monomodal Gaussian distribution of anti-a362 IgA levels were found for controls, and a bimodal distribution was found for patients with Crohn's disease. An M. paratuberculosis etiology is suggested for the 36% of patients with Crohn's disease who had an anti-a362 IgA level higher than that of controls.
Antibodies, Bacterial↗
Paratuberculosis.
Paratuberculosis (Johne's disease) is a chronic, wasting, widespread mycobacteriosis of ruminants. It involves extensive mycobacterial shedding, which accounts for the high contagiousness, and ends with a fatal enteritis. Decreases in weight, milk production, and fertility produce severe economic loss. The DNA of the etiological agent (Mycobacterium paratuberculosis) has a base composition (66 to 67% G+C) within the range of that of mycobacteria (62 to 70% G+C), a size (4.4 x 10(6) to 4.7 x 10(6) bp) larger than that of most pathogenic mycobacteria (2.0 x 10(6) to 4.2 x 10(6) bp), and a high relatedness (> 90%) to Mycobacterium avium DNA. However, the DNAs of the two organisms can be distinguished by restriction fragment length polymorphism analysis. M. paratuberculosis genes coding for a transposase, a cell wall-associated protein (P34), and two heat shock proteins have been cloned and sequenced. Nucleic acid probes (two of which are species specific) are used, after PCR amplification, for M. paratuberculosis identification in stools and milk. As in leprosy, with disease progression, cellular immune reactions decrease and humoral immune reactions increase. Cutaneous testing with sensitins, lymphocyte proliferation assays, and cytokine tests are used to monitor cellular immune reactions in paratuberculosis, but these tests lack specificity. Complement fixation, immunodiffusion, and enzymometric tests based on antibodies to M. paratuberculosis extracts, to mycobacterial antigen complex A36, to glycolipids, and to proteins help identify affected cattle but are not species specific. The carboxyl-terminal portion of the 34-kDa cell wall-associated A36 protein (P34) carries species-specific B-cell epitopes and is the basis for an enzyme-linked immunosorbent assay. Diagnostic tests for paratuberculosis are also used in Crohn's disease, a chronic human ileitis mimicking Johne's disease, in which isolates identified as M. paratuberculosis have been found.
Amino Acid Sequence↗
Development of species-specific enzyme-linked immunosorbent assay for diagnosis of Johne's disease in cattle.
The previously described (M. De Kesel, P. Gilot, M.-C. Misonne, M. Coene, and C. Cocito, J. Clin. Microbiol., 31:947-954, 1993) a362 recombinant polypeptide of Mycobacterium paratuberculosis was used as reagent for an enzyme-linked immunosorbent assay (ELISA). This ELISA, which is endowed with species specificity with respect to the other mycobacteria, was applied to the analysis of bovine paratuberculosis (Johne's disease), an endemic mycobacteriosis of cattle caused by M. paratuberculosis. The distribution of anti-a362 antibodies in the cattle population was analyzed by a computer program (mixture population model) to determine a cutoff value for the test. The prevalence of a362 seropositivity in the Belgian bovine population was estimated to be 12%. The sensitivity of the a362 assay was 70%, as determined with reference sera from the U.S. National Repository of Paratuberculosis Specimens. Some 40% of the animals in the herds with paratuberculosis analyzed were found to be positive by the a362 assay. The latter proved to be 95% specific with respect to both healthy and tuberculous cattle.
Animals↗
Thermostable macromolecular antigens from mycobacteria.
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Animals↗
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↗
Isolation and sequencing of the gene coding for an antigenic 34-kilodalton protein of Mycobacterium paratuberculosis.
The gene coding for an antigenic 34-kDa protein of Mycobacterium paratuberculosis was isolated and sequenced. The 897-bp open reading frame coded for a novel protein containing specific B epitopes. The occurrence of well-defined hydrophobic and hydrophilic portions suggests the wall location of the protein.
Amino Acid Sequence↗
Cloning and expression of portions of the 34-kilodalton-protein gene of Mycobacterium paratuberculosis: its application to serological analysis of Johne's disease.
Paratuberculosis (Johne's disease), an endemic mycobacteriosis of cattle that is caused by Mycobacterium paratuberculosis, is characterized by incoercible diarrhea and fecal shedding of bacteria. The present work aimed at developing a specific serological test for this disease. We have recently shown that a 34-kDa protein belonging to the major antigen complex A36 of M. paratuberculosis is immunodominant and contains epitopes specific with respect to all mycobacteria tested, including Mycobacterium bovis and the closely related species Mycobacterium avium. From a lambda gt11 genomic library of M. paratuberculosis, three portions of the gene coding for this 34-kDa protein have been isolated. Two of them expressed cross-reacting mycobacterial epitopes. One portion (in clone a362) expressed a polypeptide which cross-reacted with all tested M. paratuberculosis strains but not with 20 other bacteria tested, including many strains of the M. avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum group. The occurrence at the M. paratuberculosis surface of epitopes corresponding to the a362 polypeptide was shown by immune electron microscopy. The recombinant a362 polypeptide was used as reagent for an enzyme-linked immunoassay for paratuberculosis. This assay correctly diagnosed all the tested blood samples from infected cattle at all stages of the disease.
Animals↗
Composition and immunological properties of the protein fraction of A36, a major antigen complex of Mycobacterium paratuberculosis.
TMA (thermostable macromolecular antigens) are major mycobacterial complexes present in all mycobacteria. We have purified A36, the TMA complex of M. paratuberculosis, the etiological agent of paratuberculosis (Johne's disease), and shown by the immune electron microscopy approach its presentation at the cell surface. The immunodominance of the A36 complex in Johne's disease was suggested by comparative ELISA analysis of infected bovine sera, using either A36 or M. paratuberculosis total soluble sonicate as antigens. The cross-reactivity of TMA complexes from different mycobacteria was evaluated by immunoenzymometric measurements. Percentage of shared epitopes was high for the couple M. paratuberculosis-M. avium, and somewhat lower for the couple M. paratuberculosis.-M. bovis. Immunological kinship between M. paratuberculosis and M. leprae was suggested by the finding that out of eleven anti-M. leprae monoclonals, four cross-reacted with A36 proteins. The specificity missing at the level of the whole A36 complex was sought at the level of its protein components. Comparative immunoblot analysis of electrophoresed A36 proteins indicated three of them to contain epitopes not shared by M. bovis proteins, and one of them to contain epitopes specific with respect to M. avium, M. bovis and M. phlei. The latter component, a 34-kDa protein, could be an ideal reagent for a serological test for Johne's disease, being immunodominant in infected cattle and endowed with species-specific epitopes.
Animals↗
Induction of cellular immune reactions by A36, an antigen complex of Mycobacterium paratuberculosis: comparison of A36 and johnin components.
Paratuberculosis (Johne's disease) is a chronic enteritis syndrome of ruminants, which is due to infection by Mycobacterium paratuberculosis. Cutaneous testing with proteins extracted from a mycobacterial culture fluid (johnin-PPD) is currently used to evaluate the cellular immune status. We have compared the components of johnin-PPD with those of the A36 complex, a thermostable macromolecular antigen (TMA) present in the cytoplasm and associated with the cell wall of M. paratuberculosis. The presence in the johnin-PPD of fifteen A36 components has been shown by Western blotting. Moreover, monoclonal antibodies, which bind respectively to the 65-kDa M. leprae heat shock protein, the 28-kDa M. leprae superoxide dismutase, and M. tuberculosis lipoarabinomannan, recognized components of the johnin-PPD. The ability of A36 to trigger delayed hypersensitivity reactions in sensitized rabbits, and to induce the proliferation of T lymphocytes from the lymph nodes of A36-sensitized mice, matched that of johnin-PPD. The homology levels of T epitopes between A36 and the TMA complexes of M. phlei, M. bovis, M. tuberculosis and M. avium were estimated, in a lymphoproliferation assay, to be 51, 52, 59 and 94% respectively. A strong cross-reactivity of A36 with an M. leprae sonicate was also observed by cutaneous testing. The A36 components within the 45.2-26.8-kDa and the 21.6-19.8-kDa ranges were proved to induce the proliferation of T lymphocytes from sensitized mice. This work supports the possible use of the A36 complex, and of some of its components, for cutaneous tests and lymphocyte proliferation assays, in order to monitor cellular immunity in Johne's disease.
Animals↗
Influence of vasopressin on learning and memory.
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Aged↗