Publications by authors named "María I Romano"

10 Publications

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Development of a lateral flow immunochromatography test for the rapid detection of bovine tuberculosis.

J Immunol Methods 2021 Apr 13;491:112941. Epub 2020 Dec 13.

Instituto de Agrobiotecnología y Biología Molecular (IABIMO), CONICET-INTA, Buenos Aires, Argentina.

Detection of specific antibodies would be a useful test strategy for bovine tuberculosis (bTB) as a complement to the single skin test. We developed a lateral flow immunochromatography (LFIC) test for rapid bTB detection based on the use of a conjugate of gold nanoparticles with a recombinant G protein. After evaluating 3 Mycobacterium bovis (MB) antigens: ESAT-6, CFP-10 and MPB83 for the control line, we selected MPB83 given it was the most specific. The performance of the test was analyzed with 820 bovine sera, 40 sera corresponding to healthy animals, 5 sera from animals infected with Mycobacterium avium subsp. paratuberculosis (MAP) and 775 sera of animals from herds with bTB. All these sera were also submitted to a validated bTB-ELISA using whole-cell antigen from MB. From the 775 sera of animals from herds with bTB, 87 sera were positive by the bTB-ELISA, 45 were positive by LFIC and only 5 animals were positives by skin test (TST). To confirm bTB infection in the group of TST (-), bTB-ELISA (+) and LFIC (+) animals, we performed postmortem examination in 15 randomly selected animals. Macroscopically, these 15 animals had numerous small and large yellow-white granulomas, characteristic of bTB, and the infection was subsequently confirmed by PCR in these tissues with lesions (gold standard). No false positive test result was detected with the developed LFIC either with the sera from healthy animals or from animals infected with MAP demonstrating that it can be a useful technique for the rapid identification of animals infected with bTB.
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http://dx.doi.org/10.1016/j.jim.2020.112941DOI Listing
April 2021

[Production and evaluation of a purified protein derivative from an Argentine strain of Mycobacterium avium subsp. paratuberculosis].

Rev Argent Microbiol 2012 Jul-Sep;44(3):155-64

Instituto de Biotecnología, CICVyA-INTA Castelar, Dr. Nicolás Repetto y De Los Reseros S/N, (1686) Hurlingham, Buenos Aires, Argentina.

Purified Protein Derivatives (PPDs) are non-defined antigens prepared from mycobacteria cultures. They are usually employed to evaluate the specific cellular immune response both in animals and humans. Bovine and avian PPDs are usually employed as antigens in mycobacterial infections such as tuberculosis and paratuberculosis. Nevertheless, PPD from Mycobacterium avium subsp. paratuberculosis, (PPDj) is neither commonly used nor frequently available. However, PPD from Mycobacterium avium subsp. avium is in fact used. We aimed to obtain and evaluate the performance of a PPDj from a local isolate of MAP using the ãInterferon-release assay. The stimulation of ãInterferon-release was significantly different between infected and control cattle when this antigen, named PPDj-IB, was used. Stimulation in the infected animals was similar with both antigens (PPDa and PPDj-IB). However, some animals were positively stimulated with PPDj-IB and not with PPDa. We demonstrated by Western blot that two antigenic molecules, lipoarabinoman and APA/ModD antigen were differentially represented in both PPDs. This could explain the difference in stimulation induction of yIFN observed at individual level. Although PPDj-IB could not improve PPDa performance, we could easily produce an effective purified protein derivative for in vitro assays.
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January 2013

Apa antigen of Mycobacterium avium subsp. paratuberculosis as a target for species-specific immunodetection of the bacteria in infected tissues of cattle with paratuberculosis.

Vet Immunol Immunopathol 2011 Sep 23;143(1-2):75-82. Epub 2011 Jun 23.

Laboratory of Biology of Recognition, Universidade Estadual do Norte Fluminense, Campos, 28013-602 Rio de Janeiro, Brazil.

Comparative genomics of Mycobacterium spp. have revealed conservative genes and respective proteins differently expressed in mycobacteria that could be used as targets for the species-specific immunodiagnostics. The alanine and proline-rich antigen Apa is a mycobacterial protein that present significant variability in primary sequence length and composition between members of M. avium and M. tuberculosis complexes. In this study, the recombinant Apa protein encoded by the MAP1569/ModD gene of M. avium subsp. paratuberculosis (Map) was used to generate a panel of monoclonal antibodies which were shown to recognize the most important veterinary pathogens of the M. avium complex, specifically Map and M. avium subsp. hominissuis, and which did not cross-react with M. bovis or M. tuberculosis. The produced antibodies were demonstrated to be a useful tool for the species-specific immunofluorescence or immunohistochemical detection of Map in experimentally infected cell cultures or intestinal tissues from cattle with bovine paratuberculosis and, additionally, they may be employed for the discrimination of pathogenic M. avium subspecies via Western blotting.
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http://dx.doi.org/10.1016/j.vetimm.2011.06.026DOI Listing
September 2011

Tuberculous meningitis: protracted course and clinical response to interferon-gamma.

Lancet Infect Dis 2007 Mar;7(3):225-32

Royal Liverpool Children's NHS Trust, Liverpool, UK.

A 12-year-old girl with protracted tuberculous meningitis received standard chemotherapy and dexamethasone and had a progressive cerebrospinal fluid neutrophilia, raised protein and depressed glucose levels. Her temperature was raised for 5 months until a second course of dexamethasone was given. At week 15, multiple tuberculomas and hydrocephalus were detected followed by acute hydrocephalus (week 58), which required a ventricular-peritoneal shunt. Tuberculomas resolved after a second course of dexamethasone but recurred 15 months later. Immunological investigations were normal including integrity of the type 1 cytokine pathway. From month 24, interferon-gamma was given subcutaneously (initially 50 microg/m(2)) and continued for 19 months. Within 2 weeks she responded clinically followed by a reduction in inflammatory signs on magnetic resonance imaging scan (but not in the tuberculomas). At month 44, when chemotherapy was stopped, the cerebrospinal fluid/serum albumin quotient was 57x10(-3) (normal <6.0x10(-3)), which supports continuing major impairment of the blood-brain barrier. Gene expression in peripheral blood mononuclear cells before and during treatment with interferon-gamma, assessed by gene array analysis, showed reduction in a number of cytokine and chemokine genes. The response to interferon-gamma might have been secondary to downregulation of certain cytokine and chemokine genes.
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http://dx.doi.org/10.1016/S1473-3099(07)70054-3DOI Listing
March 2007

Identification of genetic markers for Mycobacterium pinnipedii through genome analysis.

FEMS Microbiol Lett 2005 Jul;248(2):147-52

Institute of Biotechnology, CICVyA-INTA, 1712 Castelar, Argentina.

Tuberculosis in seals is caused by Mycobacterium pinnipedii, a member of the Mycobacterium tuberculosis complex. In this study, we evaluated the extent of genetic variability among Mycobacterium bovis and M. pinnipedii by microarray-based comparative genomics. We identified two deletions that are exclusive to M. pinnipedii: PiD1 that removes the orthologues of the M. tuberculosis genes Rv3530c and Rv3531c, and PiD2 that encompasses genes Rv1977 and Rv1978. Interestingly, a deletion overlapping the previously described RD2 region was identified in some isolates of Mycobacterium microti and further characterised.
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http://dx.doi.org/10.1016/j.femsle.2005.05.034DOI Listing
July 2005

Use of touch-down polymerase chain reaction to enhance the sensitivity of Mycobacterium bovis detection.

J Vet Diagn Invest 2005 May;17(3):232-8

Instituto de Biotecnología, CICVyA/INTA, Los Reseros y las Cabañas, 1712 Castelar, Argentina.

The confirmatory diagnosis of Mycobacterium bovis (M. bovis) in animal samples is carried out by culture in Stonebrink media. However, culture is very slow because of the extremely long duplication time of the bacillus and difficult because of the scarcity of bacilli in diagnostic samples. This study describes the development of a single-tube touch-down polymerase chain reaction (PCR) protocol for the detection of M. bovis using primers that target the IS6110 element. Spiked water and milk as well as routine diagnostic samples (milk and nasal swabs) from M. bovis-positive cattle were tested. This protocol allows the rapid and sensitive detection of M. bovis in bovine samples by enhancing the sensitivity of standard PCR amplification.
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http://dx.doi.org/10.1177/104063870501700303DOI Listing
May 2005

Microarray analysis of Mycobacterium microti reveals deletion of genes encoding PE-PPE proteins and ESAT-6 family antigens.

Tuberculosis (Edinb) 2004 ;84(3-4):159-66

Veterinary Laboratories Agency (Weybridge), Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, UK.

Mycobacterium microti is the agent of tuberculosis in wild voles and has been used as a live vaccine against tuberculosis in man and cattle. To explore the M. microti genome in greater detail, we used a M. tuberculosis H37Rv genomic DNA microarray to detect gene deletions among M. microti isolates. A number of deletions were identified that correlated with those described previously (Infect. Immun. 70 (2002) 5568) but a novel M. microti deletion was also found (MiD4) which removes 5 genes that code for ESAT-6 family antigens and PE-PPE proteins. Southern blot experiments showed that this region was also deleted from M. pinnipedii, a mycobacterium isolated from seals that is closely related to M. microti. Genes encoding ESAT-6 antigens and PE-PPE proteins appear to be frequently deleted from M. microti, and the implications of this are discussed.
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http://dx.doi.org/10.1016/j.tube.2003.12.002DOI Listing
November 2004

The knockout of the lprG-Rv1410 operon produces strong attenuation of Mycobacterium tuberculosis.

Microbes Infect 2004 Feb;6(2):182-7

Institute of Biotechnology, CICVyA-INTA, Los Reseros y Las Cabañas, 1712 Castelar, Argentina.

P27 lipoprotein was previously described as an antigen in the Mycobacterium tuberculosis complex, encoded by the lprG gene, also named Rv1411 in the TubercuList (http://genolist.pasteur.fr/TubercuList) gene bank. It forms an operon with Rv1410 that encodes for an efflux pump, P55. A mutant of the H37Rv strain of M. tuberculosis not producing P27 (strain DeltaP27) was obtained by two-step mutagenesis using the counterselectable marker sacB and a thermosensitive origin of replication in the shuttle plasmid pPR27. By RT-PCR, we observed no lprG or Rv1410 mRNA in the DeltaP27 mutant strain compared with the wild type and complemented strains. Western blot experiments using anti-P27 polyclonal sera showed that the P27 protein was present both in the parental and in a complemented strain, in which the entire lprG-Rv1410 operon was reintroduced, but absent in the mutant strain. The three strains showed similar growth kinetics and characteristics in culture broth. To study the effect of the lprG mutation on M. tuberculosis virulence, BALB/c mice were inoculated to determine bacterial loads in spleens. At days 15 and 35 after infection, decreases of 1.5 and 2.5 logs in the bacterial load were found, respectively, in animals inoculated with the DeltaP27 mutant strain or with the wild type. This attenuation was reverted in the complemented strain. These results demonstrated that lprG gene is required for growth of M. tuberculosis in immunocompetent mice. The reversion of attenuation in the complemented strain indicates that the attenuated phenotype resulted from disruption of the lprG-Rv1410 operon.
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http://dx.doi.org/10.1016/j.micinf.2003.10.010DOI Listing
February 2004

Negative transcriptional regulation of the mce3 operon in Mycobacterium tuberculosis.

Microbiology (Reading) 2002 Oct;148(Pt 10):2997-3006

Institute of Biotechnology1, Institute of Pathobiology2, Institute of Virology3, CICVyA/INTA, 1712 Castelar, Argentina.

mce3 is one of the four mce operons in Mycobacterium tuberculosis that encode exported proteins with a probable role in the virulence of this bacterium. Upstream of mce3 there is a putative regulatory gene (Rv1963) that harbours a double tetR-family signature. To study the role of this putative regulatory gene in the transcriptional regulation of the mce3 operon, Mycobacterium smegmatis mc(2)155 and M. tuberculosis H37Rv strains that harboured gene fusions between the mce3 promoter region and the Escherichia coli lacZ gene, either containing or not containing the Rv1963 gene, were used. The presence of the Rv1963 gene in the strains greatly reduced beta-galactosidase activity, suggesting that the Rv1963-encoded protein is a transcriptional repressor of the mce3 operon. Expression of mce3 by recombinant M. tuberculosis was increased when it was grown in a macrophage-like cell line (J774), compared to the level of expression seen when the recombinant bacterium was grown under in vitro conditions. However, no lifting of repression was induced. The mce3 promoter was defined by deletion and cloning of the Rv1963-Rv1964 intergenic region in a 200 bp DNA fragment harbouring the region upstream of the Rv1964 start codon. Gel-shift experiments determined that the Rv1963-binding site was located in this region. These results indicate that the mce3 operon is transcriptionally regulated and that under certain, unknown, conditions repression of gene expression could be lifted.
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http://dx.doi.org/10.1099/00221287-148-10-2997DOI Listing
October 2002

A 12.7 kb fragment of the Mycobacterium tuberculosis genome is not present in Mycobacterium bovis.

Microbiology (Reading) 1999 Apr;145 ( Pt 4):893-897

Instituto de Biotecnología, Centro de Investigaciones en Ciencias Veterinarias, Instituto Nacional de Tecnología Agropecuaria, 1708 Moron, Argentina.

Southern blotting, sequence analysis and PCR experiments showed that Mycobacterium bovis and Mycobacterium bovis BCG lack a 12.7 kb fragment present in the genome of Mycobacterium tuberculosis. This region is 337 bp downstream of the RD2 region, which was previously described as being absent from some M. bovis BCG strains. The 12.7 kb fragment should be useful as a target for a PCR test to differentiate M. tuberculosis and M. bovis. An analysis of the 12.7 kb region suggests that it represents a deletion in M. bovis rather than an insertion in M. tuberculosis. The deletion removes most of the mce-3 operon, one of four highly related operons which may be involved in cell entry, and therefore it may contribute to differences in virulence or host range in the two species.
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http://dx.doi.org/10.1099/13500872-145-4-893DOI Listing
April 1999
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