[Application of polymerase chain reaction (PCR) in the detection of mycobacteria in milk].
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Animals↗
Biomedical subjects
M I Romano
Publications and source records attributed to M I Romano.
[Characterization of mycobacterial antigens of sea lions].
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Animals↗
[Application of spoligotyping to the analysis of transmission of Mycobacterium bovis in rodeo cattle and goats].
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Animals↗
Molecular epidemiology of Mycobacterium bovis isolates from South America.
One hundred seventy-eight isolates of Mycobacterium bovis were subjected to DNA restriction fragment length polymorphism (RFLP) analysis, using the direct repeat element (DR) and the polymorphic GC-rich repeat sequence (PGRS) as probes. By combining the patterns generated by the two repeat DNA elements, 93 different patterns were observed. One hundred-one isolates were grouped in clusters, which include 25 different clusters. One pattern was the most frequently observed, clustering 18.5% of isolates. It was only found in the Center and northeast regions of Argentina and in one isolate from Paraguay. The isolates from Brazil analyzed here presented exclusive patterns (only found in a particular region). The number of exclusive patterns was high in all argentine regions: northeast 78%, center 81%, and Buenos Aires 81%.
Animals↗
An mpb-64 flanking sequence specific for Mycobacterium bovis.
A clone carrying a plasmid with the mpb-64 gene and 3' flanking sequences (plasmid pMBA122) was detected during the screening of a Mycobacterium bovis genomic library with sera from infected cattle. When the pMBA122 insert was used as a probe in Southern blots against PvuII-digested mycobacterial DNA, it distinguished the different M. tuberculosis complex species. This probe hybridized with a 7-kb band in M. tuberculosis, a 5-kb band in M. bovis and a 3-kb band in M. tuberculosis complex strains from wild seals. Smal genomic digestions enabled us to locate this polymorphic region downstream of the mpb-64 gene. In order to clone this particular region, we designed a pair of PCR primers. Unexpectedly, these primers amplified only M. bovis DNA; no amplification was seen in M. tuberculosis DNA. When the annealing temperature was lowered from 70 to 55 degrees C, an amplification product of the same size was obtained with M. tuberculosis. This product was cloned and sequenced, and showed partial homology to the M. bovis amplified fragment. Therefore, this region comprises M. bovis sequences with a lower homology with M. tuberculosis than other compared sequences. This suggests that a more precise differentiation method at the species level for the M. tuberculosis complex could be achieved using PCR directed to this region.
Animals↗
A novel 27 kDa lipoprotein antigen from Mycobacterium bovis.
A novel Mycobacterium bovis antigen was identified from an expression library using sera from naturally infected cattle. The Escherichia coli recombinant clone expressed a 27 kDa protein, named P27. A rabbit serum against the recombinant antigen recognized a protein of 27 kDa in cellular extracts from M. bovis and M. tuberculosis. No protein was recognized in the culture supernatant. Sequence analysis indicated that P27 has a molecular mass of 24 kDa, showing a characteristic signal sequence for lipoprotein modification (a signal peptidase type II site). The gene is identical to a gene identified in the M. tuberculosis genome sequencing project. Cellular fractionation experiments suggested that P27 is an integral membrane protein. The antigen was recognized by individual sera and peripheral blood mononuclear cells (PBMC) from diseased cattle. PCR experiments with specific primers directed to the P27 structural gene indicated that it is only present in the M. tuberculosis species complex. In conclusion, a novel immunogenic lipoprotein in M. bovis/M. tuberculosis has been identified. The results presented here and elsewhere suggest that mycobacterial lipoproteins should be considered in the design of new recombinant vaccines and diagnostic methods.
Amino Acid Sequence↗
Comparison of different genetic markers for molecular epidemiology of bovine tuberculosis.
In the present study three different genetic markers were used in an RFLP study to differentiate Mycobacterium bovis (M. bovis) isolated in Argentina. The markers were: the insertion sequence IS6110, the direct repeat (DR) sequence flanking IS6110, and a polymorphic GC-rich repetitive sequence (PGRS), called pMBA2. Two restriction enzymes were used, PvuII for IS6110 and DR (DR/PvuII) and AluI for DR (DR/AluI) and pMBA2. DNA from 85 of M. bovis isolates was digested with PvuII and hybridized with IS6110 and Dr. IS6110 was not useful to differentiate M. bovis because most of the isolates contain a single monomorphic copy. The use of DR allowed a limited degree of differentiation. DNA from 44 of these isolates was also digested with AluI and hybridized with DR and pMBA2. In this condition these probes differentiated the isolates in many different RFLP types. By combining the patterns generated with DR/AluI and pMBA2 it was possible to increase the differentiation up to obtain 30 different RFLP types and 54% of the isolates were differentiated because they showed a unique pattern. Six isolates of M. bovis involved in two different outbreaks of bovine tuberculosis were correctly identified. Thus, DR and pMBA2 could be, at the moment, the probes of choice for comparisons of M. bovis isolates in different regions and for epidemiological surveillance of bovine tuberculosis.
Animals↗
Tuberculosis in sea lions and fur seals from the south-western Atlantic coast.
Diverse pathological conditions causing the strandings and/or deaths of several species of sea lions and seals on the northern coast of the province of Buenos Aires are being studied. Tuberculosis was diagnosed in six cases of strandings, involving two otariid seal species (one Otaria flavescens and five Arctocephalus australis), between March 1989 and December 1992. Necropsies were performed on all six cases. Granulomatous lesions were observed in the prescapular and hepatic lymph nodes. Lesions were also seen in the lungs, pleura, liver, spleen and peritoneum. Bacteriological isolation was attempted from all the samples. The isolates were identified as belonging to the Mycobacterium tuberculosis complex. Some showed characteristics consistent with M. bovis, whereas others demonstrated properties of M. tuberculosis. Genomic deoxyribonucleic acid (DNA) from these strains was analysed by restriction fragment length polymorphism (RFLP), using IS6110, a genetic marker found only in the Mycobacterium tuberculosis complex. Using the IS6110 probe, similar fingerprints were obtained, suggesting a common source of infection. However, the pattern of DNA differed from DNA patterns of M. bovis isolated from humans and cattle in Argentina, which generally contain a unique 1.9 kbp band. These results suggest that mycobacteria isolated from wild seals form a different grouping inside the M. tuberculosis complex. This is the first time that tuberculosis has been detected in wild seals from the south-western Atlantic coast.
Animals↗
Multidrug resistant tuberculosis outbreak in Buenos Aires. DNA fingerprinting analysis of isolates.
In order to determine the possible relationship among HIV patients coinfected with multidrug resistant tuberculosis strains who had been receiving clinical assistance in our Hospital, clinical and epidemiological information from 28 patients was collected. DNA fingerprinting by restriction fragment length polymorphism (RFLP) pattern was performed on the mycobacterial isolates from these patients, using the restriction enzyme Pvull and IS 6110 as genetic marker. A unique RFLP pattern was found in 10 isolates from 10 different patients who had a disease caused by a single strain. Our findings confirm RFLP as a reliable and useful tool to analyze TB transmission.
Acquired Immunodeficiency Syndrome↗
Genetic characterization of mycobacteria from South American wild seals.
Tuberculosis has been recently diagnosed in four wild seals found stranded in the Atlantic coast of Argentina. By bacteriological studies and IS6110 hybridization, these isolates were characterized as belonging to the Mycobacterium tuberculosis complex. A genetic characterization using RFLP (Restriction fragment length polymorphism) and a species-specific probe of M. tuberculosis, called mtp40, showed hybridization with this probe on a single band. A similar band was also found in M. tuberculosis H37Rv. This showed a relationship between M. tuberculosis and the wild seal isolates. However these would also seem to belong to a different genetic group in the M. tuberculosis complex, since they do not grow on glycerol-egg containing medium (Lowenstein-Jensen) as typical M. tuberculosis strains usually do. Repeated sequences pMBA2, pTNB12, DR and IS6110 were used as probes to evaluate the epidemiological relationships between the 4 cases of tuberculosis. A low degree of polymorphism was observed, that suggested that these isolates were epidemiologically related.
Animals↗
Cloning of a novel polymorphic GC-rich repetitive DNA from Mycobacterium bovis.
A repetitive DNA from a wild-type Mycobacterium bovis isolate was cloned and characterized. The repeated segment was present in M. bovis and M. tuberculosis but was absent from the six other mycobacteria tested. Sequence analysis demonstrated that this repetitive element belonged to the polymorphic GC-rich repeat sequence type, a family of interspersed repeated DNA. This fragment, when used as a probe in restriction fragment length polymorphism analyses, was able to detect polymorphism in M. bovis genotypes that went undetected when the established IS6110 was used as a probe. This repetitive element should be useful in epidemiological studies of bovine tuberculosis.
Base Sequence↗
Characterization of a novel Mycobacterium bovis secreted antigen containing PGLTS repeats.
Serum from naturally infected cattle was used to identify a novel Mycobacterium bovis antigen from an expression library. The first recombinant product identified was a fusion protein with lacZ (55 kDa). A clone containing the whole gene was also obtained. This clone expressed a 38-kDa protein. A rabbit serum against the recombinant antigen reacts in M. bovis supernatants with two proteins of 36 and 34 kDa. The new protein was called P36/P34. The gene cloned has a deduced amino acid sequence with a predicted molecular mass of 28 kDa, showing a characteristic signal sequence for exportation. The protein bears partial homology to a 28-kDa protein from M. leprae. An interesting feature of the P36/P34 sequence is that it contains several PGLTS repeats, which are not present in the M. leprae protein. Antigenic determinants seem also to be conserved between the two proteins because sera from leprosy patients recognized the recombinant M. bovis protein. The discrepancy among the molecular mass deduced from the sequence (28 kDa), that of the recombinant protein in Escherichia coli (38 kDa), and that of the native protein in M. bovis (36 and 34 kDa) could be attributed to posttranslational modifications or to the high proline content that may alter the migration properties of the protein. This antigen seems to be immunodominant during bovine tuberculosis, because 8 of 9 serum specimens from diseased cattle are reactive. The homology among the M. leprae 28-kDa protein, the protein described in this article, and a recently described M. tuberculosis protein suggests the existence of a new protein family in mycobacteria.
Amino Acid Sequence↗
Relationship between the level of c-myc mRNA and histologic aggressiveness in thyroid tumors.
We investigated the relationship between the level of c-myc mRNA and histologic aggressiveness in thyroid tumors obtained at surgery. In thyroid carcinomas, there was a positive correlation between these two parameters, while in benign tumors there was no correlation between cellularity and the expression of the proto-oncogene c-myc. These results might be useful in the prognosis of thyroid tumors and consequently helpful in the management of the patient.
Carcinoma, Papillary↗
Relationship between release of LH and incorporation of tritiated thymidine in the anterior pituitary gland of the castrated male rat: effect of LHRH and its highly active analogue buserelin.
Castration of the adult male rat significantly (P less than 0.01) increased the concentration of LH in serum and the incorporation of (3H) thymidine into the pituitary DNA. The administration of a single dose of LHRH or its analogue buserelin stimulated the release of LH but it did not modify (3H) thymidine incorporation. When multiple doses of LHRH or buserelin were injected, there was a significant (P less than 0.01) inhibition of LH release and also the incorporation of (3H) thymidine into the DNA of the anterior pituitary gland was significantly (P less than 0.01) diminished. These observations are compatible with the idea of the close relationship between hormonal release and DNA synthesis in the anterior pituitary gland of the rat.
Animals↗
Prolactin release, oestrogens and proliferation of prolactin-secreting cells in the anterior pituitary gland of adult male rats.
Relationships among the release of prolactin, the effect of oestrogens and the proliferation of prolactin-secreting cells were studied under several experimental conditions. Administration of sulpiride or oestradiol released prolactin and stimulated cell proliferation in the anterior pituitary gland of adult male rats. Clomiphene completely abolished the rise in cell proliferation, but did not interfere with the sulpiride-induced release of prolactin. Treatment with oestradiol plus sulpiride significantly increased serum prolactin concentrations and the mitotic index compared with the sum of the stimulation produced by both drugs separately. Bromocriptine abolished the stimulatory effect of oestradiol on the serum prolactin concentration and on cell proliferation. In oestradiol- and/or sulpiride-treated rats, 80% of the cells in mitoses were lactotrophs. The remaining 20% did not stain with antisera against any of the pituitary hormones. The number of prolactin-secreting cells in the anterior pituitary gland significantly increased after the administration of oestradiol or sulpiride. The results demonstrate that treatment with sulpiride and/or oestradiol increases the proliferation and the number of lactotrophs in the anterior pituitary gland of the rat.
Animals↗
Relationship between release of LH and incorporation of tritiated thymidine in the anterior pituitary gland of the castrated female rat.
Castration of female rats during diestrus increases the concentration of circulating LH from days 3 and the incorporation of 3H thymidine into pituitary DNA from day 5. Both effects are completely abolished by the administration of dihydrotestosterone. Although the i.v. injection of LHRH markedly enhances the concentration of LH in serum, it does not modify the incorporation of 3H thymidine into pituitary DNA. Castration might produce a maximal stimulation in 3H thymidine incorporation and a further stimulation of LH release with LHRH is unable to enhance the incorporation of the radioactive precursor. The results suggest a relationship between LH secretion and DNA synthesis in the pituitary gland of the rat.
Animals↗
Effect of ovariectomy and clomiphene on the in-vitro incorporation of [3H]thymidine into pituitary DNA and on prolactin synthesis and release in rats.
In the anterior pituitary gland changes in prolactin synthesis and in the incorporation of [3H]thymidine into DNA are coincident under several experimental conditions. We investigated whether these changes are obligatory, thus indicating a regulatory mechanism common to the synthesis of both macromolecules. Alternatively, the parallel changes may represent similar responses to various stimuli operating through different pathways. The administration of alpha-methyl-p-tyrosine (alpha MpT) to rats stimulated the incorporation of [3H]leucine into prolactin and [3H]thymidine into DNA. When the effectiveness of oestrogen was suppressed by ovariectomy or by blockage of oestrogen receptors by the antioestrogen clomiphene, alpha MpT stimulated the synthesis of prolactin but not the incorporation of [3H]thymidine into pituitary DNA. The results clearly indicate two independent mechanisms regulating the synthesis of prolactin and DNA in the anterior pituitary gland.
Animals↗
Correlation between LH secretion in castrated rats with cellular proliferation and synthesis of DNA in the anterior pituitary gland.
The relationship between the release of LH and the synthesis of DNA was studied in the anterior pituitary gland of castrated rats. Cell types were characterized immunocytochemically. Castration significantly (P less than 0.01) increased the concentration of LH in serum (1326%) and the incorporation of [3H]thymidine into pituitary DNA (72%). This was accompanied by an increment in the activity of the enzyme DNA polymerase-alpha (58%) and in the number of mitoses (from 2 +/- 0.1/mm2 in intact rats to 21 +/- 0.8/mm2 15 days after castration). Only 20% of the mitoses found in the pituitary gland of castrated rats were positively stained with the antiserum against the beta-subunit of LH. The other 80% did not stain either with LH antiserum or with antisera against the other pituitary hormones. There was a significant (P less than 0.01) increase in the number of LH cells in castrated rats (48%). All the changes produced in the anterior pituitary gland after castration were prevented by the administration of dihydrotestosterone. The results demonstrate that a stimulation of LH release is followed by an increase of DNA synthesis and cell proliferation of gonadotrophs in the anterior pituitary gland.
Animals↗